Bifunctional stapled polypeptides and uses thereof

ABSTRACT

The invention relates to bifunctional stapled or stitched peptides comprising a targeting domain, a linker moiety, and an effector domain, that can be used to tether, or to bring into close proximity, at least two cellular entities (e.g., proteins). Certain aspects relate to bifunctional stapled or stitched peptides that bind to an effector biomolecule through the effector domain and bind to a target biomolecule through the targeting domain. Polypeptides and/or polypeptide complexes that are tethered by the bifunctional stapled or stitched peptides of the invention, where the effector polypeptide bound to the effector domain of the bifunctional stapled or stitched peptide modifies or alters the target polypeptide bound to the targeting domain of the bifunctional peptide. Uses of the inventive bifunctional stapled or stitched peptides including methods for treatment of disease (e.g., cancer, inflammatory diseases) are also provided.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/789,421, filed Oct. 20, 2017, which is a continuation of U.S.application Ser. No. 14/880,080, filed Oct. 9, 2015, which is acontinuation of U.S. application Ser. No. 13/383,881, filed Jul. 6,2012, now U.S. Pat. No. 9,163,330, which is a national stage filingunder 35 U.S.C. § 371 of international PCT application,PCT/US2010/001952, filed Jul. 13, 2010, which claims the benefit of U.S.Provisional Patent Application No. 61/225,191, filed Jul. 13, 2009, eachof which is incorporated herein by reference.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created Oct. 19, 2017 isnamed 085298_000309_SL and is 62,591 bytes in size.

BACKGROUND OF THE INVENTION

The important biological roles that peptides and proteins play ashormones, enzyme inhibitors, substrates, and neurotransmitters has ledto the use of peptides and/or peptide mimetics as therapeutic agents.The peptide's bioactive conformation, combining structural elements suchas alpha-helices, beta-sheets, turns, and/or loops, is important as itallows for selective biological recognition of receptors, enzymes, andnucleic acids, thereby influencing cell-cell communication and/orcontrolling vital cellular functions, such as metabolism, immunedefense, and cell division (Babine et al., Chem. Rev. (1997) 97:1359).Unfortunately, the utility of peptides as drugs is severely limited byseveral factors, including their rapid degradation by proteases underphysiological conditions, their poor cell permeability, and their lackof binding specificity resulting from conformational flexibility.

The alpha-helix is one of the major structural components of peptides.However, alpha-helical peptides have a propensity for unraveling andforming random coils, which are, in most cases, biologically lessactive, or even inactive, and are highly susceptible to proteolyticdegradation.

Many research groups have developed strategies for the design andsynthesis of more robust peptides as therapeutics. For example, onestrategy has been to incorporate more robust functionalities into thepeptide chain while still maintaining the peptide's unique conformationand secondary structure (see, for example, Gante, Angew. Chem. Int. Ed.Engl. (1994) 33:1699-1720; Liskamp, Recl. Trav. Chim. Pays-Bas (1994)113:1; Giannis, Angew. Chem. Int. Ed. Engl. (1993) 32:1244; Bailey,Peptide Chemistry, Wiley, New York (1990), 182; and references citedtherein). Another approach has been to stabilize the peptide viacovalent cross-links (see, for example, Phelan et al., J. Am. Chem. Soc.(1997) 119:455; Leuc et al., Proc. Natl. Acad. Sci. USA (2003) 100:11273; Bracken et al., J. Am. Chem. Soc. (1994) 116:6432; Yan et al.,Bioorg. Med. Chem. (2004) 14:1403). However, the majority of reportedapproaches involved the use of polar and/or labile cross-linking groups.

“Peptide stapling” is a term coined for a synthetic methodology used tocovalently join two olefin-containing side chains present in apolypeptide chain using an olefin metathesis reaction (J. Org. Chem.(2001) 66(16); Blackwell et al., Angew. Chem. Int. Ed. (1994) 37:3281).Stapling of a peptide using a hydrocarbon cross-linker created from anolefin metathesis reaction has bee shown to help maintain a peptide'snative conformation, particularly under physiological conditions (U.S.Pat. No. 7,192,713; Schafmeister et al., J. Am. Chem. Soc. (2000)122:5891-5892; Walensky et al., Science (2004) 305:1466-1470; each ofwhich is incorporated herein by reference). This strategy has beenapplied to the apoptosis-inducing BID-BH3 alpha-helix, resulting in ahigher suppression of malignant growth of leukemia in an animal modelcompared to the unstapled peptide (Walensky et al., Science (2004)305:1466-1470; U.S. Patent Application Publication No. 2005/02506890;U.S. Patent Application Publication No. 2006/0008848; each of which isincorporated herein by reference).

SUMMARY OF THE INVENTION

The present invention stems from the recognition of a new use forstapled or stitched peptides. Given the stability of such peptides, theymay be used as agents for recruiting proteins or other biomolecules to aparticular protein, nucleic acid, other biomolecule, cell, or organelle(i.e., tethering two cellular components together or brining them intoclose proximity). In particular, the present invention providesbifunctional peptides, one or both domains of which may be stapled orstitched. One domain of the bifunctional peptide acts as a targetingmoiety that binds to a target; the other domain acts as an effectordomain to recruit a protein or protein complex to the target. Theeffector domain typically acts on or modifies the activity of thetarget. In essence, the bifunctional peptide works to bring two proteinsor other biomolecules in close proximity to one another. The targetingdomain, the effector domain, or both domains may be stapled or stitchedto stabilize the conformation of the peptide. The two domains are linkedtogether via a linker, which may range in structure from simply acovalent bond to a bifunctional molecule to a polymeric linker.

In one aspect, the present invention provides a bifunctional peptidewherein one or both of the targeting domain and effector domain arestapled or stitched. The inventive bifunctional peptide includes atargeting domain associated with an effector domain. Each peptidecomprises 5-100 amino acids as needed to act as a ligand for a targetedprotein. The peptide may include unnatural amino acids with olefin sidechains as necessary to form a staple or stitch used to stabilize theconformation of the peptide. In certain embodiments, the stapled orstitched peptide is a helical peptide. Typically, the two domains arecovalently associated with one another through a linker; however,non-covalent associations may also be used. In certain embodiments, thebifunctional peptide is a stapled version of SAH p53-8 associated with astapled version of Bcl-9. In other embodiments, the bifunctional peptideis a stapled version of SAH p53-8 associated with Tcf4. Such inventivebifunctional peptides promote the degradation of β-catenin by recruitingE3 ubiquitin ligase to β-catenin. E3 ubiquitin ligase then catalyzes theubiquitination of β-catenin, resulting in its degradation by theproteasome.

In certain embodiments, an inventive bifunctional stapled or stitchedpeptide comprising a targeting domain, a linker, and an effector domainare the focus of the present invention. The present invention providesbifunctional stapled or stitched peptides, and methods for theirpreparation and use. The present invention also provides pharmaceuticalcompositions comprising an inventive bifunctional stapled or stitchedpeptide and a pharmaceutically acceptable excipient. In certainembodiments, the present invention provides bifunctional, alpha-helicalstapled or stitched peptides, wherein at least one of the peptides isalpha-helical and stabilized by stapling or stitching. In certainembodiments, the inventive alpha-helical peptide retains itsalpha-helical structure under physiological conditions, such as in thebody of a subject (e.g., in the gastrointestinal tract; in thebloodstream).

In certain embodiments, stapled or stitched bifunctional peptidescomprising a targeting domain, a linker, and an effector domain aregenerally arranged as follows:

wherein A and E are peptides or peptide-like; A and/or E is a stapled orstitched peptide; and L is a linker (e.g., covalent bond; polyethyleneglycol (PEG); aminohexanoic acid-based linker; poly-glycine peptide,monodispers polymer etc.), and wherein if A is a targeting domain and Eis an effector domain.

In one aspect, the present invention provides a bifunctional stapled orstitched peptide wherein one or both domains (i.e., A or E) are of theformula:

wherein L₁, L₂, R^(a), R^(b), R^(e), R^(f), R^(LL), X_(AA), s, t, q, z,j, and

are as described herein.

In another aspect, the present invention provides a bifunctionalstitched peptide wherein one or both domains are of the formula (i.e., apeptide with multiple staples):

wherein K, L₁, L₂, M, R^(a), R^(b), R^(e), R^(f), R^(KL), R^(LL),R^(LM), X_(AA), y, z, j, p, s, t, u, v, q, and

are as described herein.

The amino acid sequence of one or both of the domains may besubstantially similar to or homologous to a known peptide. In someembodiments, the targeting domain binds a protein, nucleic acid, orother biomolecule. In certain embodiments, the targeting domain bindsβ-catenin, c-Myc, Ras, or hypoxia-inducible factor. In some embodiments,the effector domain recruits an enzyme to a target molecule. In certainembodiments, the effector domain is a ligand for a ubiquitinating enzyme(e.g., E3 ubiquitin ligase), a glycosylating enzyme, a histonedeacetylase, a histone acyl transferase, a kinase, a protease, afarnesyl transferase, an acetylase, or a phosphatase.

The linker may be proteinogenic or non-proteinogenic. The linker may beas simple as a covalent bond (e.g., a carbon-carbon bond, disulfidebond, carbon-heteroatom bond, etc.), or it may be more complicated suchas a polymeric linker (e.g., polyethylene, polyethylene glycol,polyamide, polyester, etc.). In certain embodiments, the linkercomprises a monomer, dimer, or polymer of aminoalkanoic acid. In certainembodiments, the linker comprises an aminoalkanoic acid (e.g., glycine,ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid,4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments,the linker comprises a monomer, dimer, or polymer of aminohexanoic acid(Ahx). In other embodiments, the linker comprises a polyethylene glycolmoiety (PEG). In other embodiments, the linker comprises amino acids.The linker may included functionalized moieties to facilitate attachmentof a nucleophile (e.g., thiol, amino) from the peptide to the linker. Incertain embodiments, the linker includes a maleimide group. In certainembodiments, the linker includes a NHS ester. In certain embodiments,the linker includes both a NHS ester and a maleimide group.

To give but one example where a stapled bifunctional peptide may beuseful in treating or studying a disease or other biological process,consider the loss of endogenous β-catenin degradation in human cancers.To restore β-catenin degradation, an inventive bifunctional stapledpeptide is used. The bifunctional peptide includes a stapled β-cateninligand (e.g., Bcl-9 or Tcf4) associated with an E3 ubiquitin ligaseligand (SAH p53-8); thereby recruiting ubiquitination machinery to theβ-catenin to be degraded. The β-catenin is ubiquitinated by ubiquitinligase leading to its destruction in the proteasome. As will beappreciated by those of skill in this art, proteins other than β-cateninmay be targeted for ubiquitination using this approach, and/or othercellular machinery or enzymes may be recruited to the target besidesubiquitination machinery. For example, enzymes or enzyme complexes suchas kinases, phosphatases, proteases, glycosylases, ligases, acetylases,lipidases, etc. may be recruited to a targeted protein. Almost anypost-translational modification including degradation of a protein maybe promoted using the inventive bifunctional peptide. Such inventivebifunctional peptides may be used in pharmaceutical compositions totreat disease in a subject (e.g., human).

The invention also provides a system for designing and preparingbifunctional peptides. One or both domains of the bifunctional peptidemay be already known in the art. The peptide domain may then be modifiedto increase its affinity for the targeted protein. The peptide may alsobe modified to include the unnatural amino acids needed to staple orstitch the peptide. In certain embodiments, a library of peptides withvarious mutations may be screened to identify a peptide with a highaffinity for the target protein. The library may include stapled orunstapled, stitched or unstitched peptides. In certain embodiments, apeptide domain may be designed in silico using structural information ofthe target protein or of a known protein-protein interaction. Indesigning the peptide domain it may need to be determined where the oneor more staples are to be placed and/or substitution in the primarysequence to yield a better bifunctional peptide. The designed peptide(s)may be assayed for the desired activity using techniques known in theart for assessing binding affinity, functionality, stability,pharmacokinetics, etc. Once the bifunctional peptide is designed it canbe prepared using available peptide chemistry. For example, a peptidemay be synthesized using standard solid phase peptide synthesismethodology. Unnatural amino acids (e.g., S₅, R₅, S₈, R₈) as needed ordesired may be introduced into the primary sequence. The peptide oncesynthesized is associated with the other peptide, or the entirebifunctional peptide may be created at once. The peptide may be stapled,stitched, deprotected, or otherwise modified before or after it isassociated with the other peptide domain.

The inventive bifunctional peptides may be used as therapeutics as wellas research tools. In certain embodiments, the inventive bifunctionalpeptide is used in the treatment of a disease in a subject (e.g., aproliferative disease, a neurodegenerative disease). For example, theTcf4-SAH p53 peptide or the Bcl-9-SAH p53 peptide as described herein(see FIGS. 8-11; SEQ ID NO: 1-20) may be used to treat cancer in asubject. As will be appreciated by one of skill in the art, almost anydisease, disorder, or condition may be treated using the inventivebifunctional peptide. The effector and targeting domains of thebifunctional peptide may be tailored for the specific use. The inventivebifunctional peptides may also be used as research tools. For example,the bifunctional peptide may be used to probe the function of aparticular protein in a cell. Increasing the degradation will allow aresearcher to understand how a deficit of the protein affects a pathwayor cell. Promoting the phosphorylation or other secondary modificationwill allow a researcher to understand how the state of a protein affectsits role in a biological pathway or cell.

In another aspect, the invention provides a kit with the componentsnecessary for designing and preparing an inventive bifunctional peptide.The kit may include containers, enzymes, buffers, amino acids, reagents,catalysts, software, instructions, etc. needed to make and/or use aninventive bifunctional peptide.

Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5^(th) Edition, John Wiley & Sons, Inc., New York, 2001;Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., NewYork, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd)Edition, Cambridge University Press, Cambridge, 1987.

“Stapling,” “hydrocarbon-stapling” as used herein introduces into apeptide at least two moieties capable of undergoing reaction to promotecarbon-carbon bond formation that can be contacted with a reagent togenerate at least one cross-linker between the at least two moieties.Stapling provides a constraint on a secondary structure, such as analpha helix structure. The length and geometry of the cross-linker canbe optimized to improve the yield of the desired secondary structurecontent. The constraint provided can, for example, prevent the secondarystructure to unfold and/or can reinforce the shape of the secondarystructure. A secondary structure that is prevented from unfolding is,for example, more stable.

A “stapled” peptide is a peptide comprising a selected number ofstandard or non-standard amino acids, further comprising at least twomoieties capable of undergoing reaction to promote carbon-carbon bondformation, that has been contacted with a reagent to generate at leastone cross-linker between the at least two moieties, which modulates, forexample, peptide stability.

A “stitched” peptide, as used herein, is a stapled peptide comprisingmore than one, that is multiple (two, three, four, five, six, etc.)cross-linked moieties.

The compounds, proteins, or peptides of the present invention (e.g.,amino acids, and unstapled, partially stapled, and stapled peptides andproteins, and unstitched, partially stitched, and stitched peptides andproteins) may exist in particular geometric or stereoisomeric forms. Thepresent invention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)- and(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention.

Where an isomer/enantiomer is preferred, it may, in some embodiments, beprovided substantially free of the corresponding enantiomer, and mayalso be referred to as “optically enriched.” “Optically enriched,” asused herein, means that the compound is made up of a significantlygreater proportion of one enantiomer. In certain embodiments thecompound of the present invention is made up of at least about 90% byweight of a preferred enantiomer. In other embodiments the compound ismade up of at least about 95%, 98%, or 99% by weight of a preferredenantiomer. Preferred enantiomers may be isolated from racemic mixturesby any method known to those skilled in the art, including chiral highpressure liquid chromatography (HPLC) and the formation andcrystallization of chiral salts or prepared by asymmetric syntheses.See, for example, Jacques et al., Enantiomers, Racemates and Resolutions(Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725(1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, N Y,1962); Wilen, Tables of Resolving Agents and Optical Resolutions p. 268(E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972).

It will be appreciated that the compounds of the present invention, asdescribed herein, may be substituted with any number of substituents orfunctional moieties. In general, the term “substituted” whether precededby the term “optionally” or not, and substituents contained in formulasof this invention, refer to the replacement of hydrogen radicals in agiven structure with the radical of a specified substituent. When morethan one position in any given structure may be substituted with morethan one substituent selected from a specified group, the substituentmay be either the same or different at every position. As used herein,the term “substituted” is contemplated to include substitution with allpermissible substituents of organic compounds, any of the substituentsdescribed herein (for example, aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,etc.), and any combination thereof (for example, aliphaticamino,heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, acyloxy, and the like) that results in the formationof a stable moiety. The present invention contemplates any and all suchcombinations in order to arrive at a stable substituent/moiety.Additional examples of generally applicable substituents are illustratedby the specific embodiments shown in the Examples, which are describedherein. For purposes of this invention, heteroatoms such as nitrogen mayhave hydrogen substituents and/or any suitable substituent as describedherein which satisfy the valencies of the heteroatoms and results in theformation of a stable moiety.

As used herein, substituent names which end in the suffix “-ene” referto a biradical derived from the removal of two hydrogen atoms from thesubstituent. Thus, for example, acyl is acylene; alkyl is alkylene;alkeneyl is alkenylene; alkynyl is alkynylene; heteroalkyl isheteroalkylene, heteroalkenyl is heteroalkenylene, heteroalkynyl isheteroalkynylene, aryl is arylene, and heteroaryl is heteroarylene.

The term “acyl,” as used herein, refers to a group having the generalformula —C(═O)R^(A), —C(═O)OR^(A), —C(═O)—O—C(═O)R^(A), —C(═O)SR^(A),—C(═O)N(R^(A))₂, —C(═S)R^(A), —C(═S)N(R^(A))₂, and —C(═S)S(R^(A)),—C(═NR^(A))R^(A), —C(═NR^(A))OR^(A), —C(═NR^(A))SR^(A), and—C(═NR^(A))N(R^(A))₂, wherein R^(A) is hydrogen; halogen; substituted orunsubstituted hydroxyl; substituted or unsubstituted thiol; substitutedor unsubstituted amino; substituted or unsubstituted acyl, cyclic oracyclic, substituted or unsubstituted, branched or unbranched aliphatic;cyclic or acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; cyclic or acyclic, substituted or unsubstituted,branched or unbranched alkyl; cyclic or acyclic, substituted orunsubstituted, branched or unbranched alkenyl; substituted orunsubstituted alkynyl; substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy,heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, mono- or di-aliphaticamino, mono- ordi-heteroaliphaticamino, mono- or di-alkylamino, mono- ordi-heteroalkylamino, mono- or di-arylamino, or mono- ordi-heteroarylamino; or two R^(A) groups taken together form a 5- to6-membered heterocyclic ring. Exemplary acyl groups include aldehydes(—CHO), carboxylic acids (—CO₂H), ketones, acyl halides, esters, amides,imines, carbonates, carbamates, and ureas. Acyl substituents include,but are not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety (e.g., aliphatic, alkyl,alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl,oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl,thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino,heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl,aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like,each of which may or may not be further substituted).

The term “acyloxy” refers to a “substituted hydroxyl” of the formula(—OR^(i)), wherein R^(i) is an optionally substituted acyl group, asdefined herein, and the oxygen moiety is directly attached to the parentmolecule.

The term “acylene,” as used herein, refers to an acyl group having thegeneral formulae: —R⁰—(C═X¹)—R⁰—, —R⁰—X²(C═X¹)—R⁰—, or—R⁰—X²(C═X¹)X³—R⁰—, where X¹, X², and X³ is, independently, oxygen,sulfur, or NR^(r), wherein R^(r) is hydrogen or aliphatic, and R⁰ is anoptionally substituted alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, or heteroalkynylene group, as defined herein.Exemplary acylene groups wherein R⁰ is alkylene includes—(CH₂)_(T)—O(C═O)—(CH₂)_(T)—; —(CH₂)_(T)—NR^(r)(C═O)—(CH₂)_(T)—;—(CH₂)_(T)—O(C═NR^(r))—(CH₂)_(T)—;—(CH₂)_(T)—NR^(r)(C═NR^(r))—(CH₂)_(T)—; —(CH₂)_(T)—(C═O)—(CH₂)_(T)—;—(CH₂)_(T)—(C═NR^(r))—(CH₂)_(T)—; —(CH₂)_(T)—S(C═S)—(CH₂)_(T)—;—(CH₂)_(T)—NR^(r)(C═S)—(CH₂)_(T)—; —(CH₂)_(T)—S(C═NR^(r))—(CH₂)_(T)—;—(CH₂)_(T)—O(C═S)—(CH₂)_(T)—; —(CH₂)_(T)—(C═S)—(CH₂)_(T)—; or—(CH₂)_(T)—S(C═O)—(CH₂)_(T)—, and the like, which may bear one or moresubstituents; and wherein each instance of xx is, independently, aninteger between 0 to 20. Acylene groups may be cyclic or acyclic,branched or unbranched, substituted or unsubstituted. Acylenesubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “aliphatic,” as used herein, includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, and cyclic (i.e., carbocyclic) hydrocarbons, which areoptionally substituted with one or more functional groups. As will beappreciated by one of ordinary skill in the art, “aliphatic” is intendedherein to include, but is not limited to, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as usedherein, the term “alkyl” includes straight, branched and cyclic alkylgroups. An analogous convention applies to other generic terms such as“alkenyl,” “alkynyl,” and the like. Furthermore, as used herein, theterms “alkyl,” “alkenyl,” “alkynyl,” and the like encompass bothsubstituted and unsubstituted groups. In certain embodiments, as usedherein, “aliphatic” is used to indicate those aliphatic groups (cyclic,acyclic, substituted, unsubstituted, branched or unbranched) having 1-20carbon atoms. Aliphatic group substituents include, but are not limitedto, any of the substituents described herein, that result in theformation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. In some embodiments, the alkyl group employed in theinvention contains 1-20 carbon atoms. In another embodiment, the alkylgroup employed contains 1-15 carbon atoms. In another embodiment, thealkyl group employed contains 1-10 carbon atoms. In another embodiment,the alkyl group employed contains 1-8 carbon atoms. In anotherembodiment, the alkyl group employed contains 1-5 carbon atoms. Examplesof alkyl radicals include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl,iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl,n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, which maybear one or more substituents. Alkyl group substituents include, but arenot limited to, any of the substituents described herein, that result inthe formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl,alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo,imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol,halo, aliphaticamino, heteroaliphaticamino, alkylamino,heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl,aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like,each of which may or may not be further substituted).

The term “alkylene,” as used herein, refers to a biradical derived froman alkyl group, as defined herein, by removal of two hydrogen atoms.Alkylene groups may be cyclic or acyclic, branched or unbranched,substituted or unsubstituted. Alkylene group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety (e.g., aliphatic, alkyl,alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl,oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl,thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino,heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl,aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like,each of which may or may not be further substituted).

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a straight- or branched-chain hydrocarbon moiety having at leastone carbon-carbon double bond by the removal of a single hydrogen atom.In certain embodiments, the alkenyl group employed in the inventioncontains 2-20 carbon atoms. In some embodiments, the alkenyl groupemployed in the invention contains 2-15 carbon atoms. In anotherembodiment, the alkenyl group employed contains 2-10 carbon atoms. Instill other embodiments, the alkenyl group contains 2-8 carbon atoms. Inyet other embodiments, the alkenyl group contains 2-5 carbons. Alkenylgroups include, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like, which may bear one or moresubstituents. Alkenyl group substituents include, but are not limitedto, any of the substituents described herein, that result in theformation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

The term “alkenylene,” as used herein, refers to a biradical derivedfrom an alkenyl group, as defined herein, by removal of two hydrogenatoms. Alkenylene groups may be cyclic or acyclic, branched orunbranched, substituted or unsubstituted. Alkenylene group substituentsinclude, but are not limited to, any of the substituents describedherein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “alkynyl,” as used herein, refers to a monovalent group derivedfrom a straight- or branched-chain hydrocarbon having at least onecarbon-carbon triple bond by the removal of a single hydrogen atom. Incertain embodiments, the alkynyl group employed in the inventioncontains 2-20 carbon atoms. In some embodiments, the alkynyl groupemployed in the invention contains 2-15 carbon atoms. In anotherembodiment, the alkynyl group employed contains 2-10 carbon atoms. Instill other embodiments, the alkynyl group contains 2-8 carbon atoms. Instill other embodiments, the alkynyl group contains 2-5 carbon atoms.Representative alkynyl groups include, but are not limited to, ethynyl,2-propynyl (propargyl), 1-propynyl, and the like, which may bear one ormore substituents. Alkynyl group substituents include, but are notlimited to, any of the substituents described herein, that result in theformation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

The term “alkynylene,” as used herein, refers to a biradical derivedfrom an alkynylene group, as defined herein, by removal of two hydrogenatoms. Alkynylene groups may be cyclic or acyclic, branched orunbranched, substituted or unsubstituted. Alkynylene group substituentsinclude, but are not limited to, any of the substituents describedherein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “amino,” as used herein, refers to a group of the formula(—NH₂). A “substituted amino” refers either to a mono-substituted amine(—NHR^(h)) of a disubstituted amine (—NR^(h) ₂), wherein the R^(h)substituent is any substituent as described herein that results in theformation of a stable moiety (e.g., a suitable amino protecting group;aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, amino, nitro, hydroxyl, thiol, halo, aliphaticamino,heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, acyloxy, and the like, each of which may or may not befurther substituted). In certain embodiments, the R^(h) substituents ofthe disubstituted amino group (—NR^(h) ₂) form a 5- to 6-memberedheterocyclic ring.

The term “alkoxy” refers to a “substituted hydroxyl” of the formula(—OR^(i)), wherein R^(i) is an optionally substituted alkyl group, asdefined herein, and the oxygen moiety is directly attached to the parentmolecule.

The term “alkylthioxy” refers to a “substituted thiol” of the formula(—SR^(r)), wherein R^(r) is an optionally substituted alkyl group, asdefined herein, and the sulfur moiety is directly attached to the parentmolecule.

The term “alkylamino” refers to a “substituted amino” of the formula(—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted alkyl group, as defined herein, and the nitrogenmoiety is directly attached to the parent molecule.

The term “aryl,” as used herein, refer to stable aromatic mono- orpolycyclic ring system having 3-20 ring atoms, of which all the ringatoms are carbon, and which may be substituted or unsubstituted. Incertain embodiments of the present invention, “aryl” refers to a mono,bi, or tricyclic C₄-C₂₀ aromatic ring system having one, two, or threearomatic rings which include, but not limited to, phenyl, biphenyl,naphthyl, and the like, which may bear one or more substituents. Arylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “arylene,” as used herein refers to an aryl biradical derivedfrom an aryl group, as defined herein, by removal of two hydrogen atoms.Arylene groups may be substituted or unsubstituted. Arylene groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).Additionally, arylene groups may be incorporated as a linker group intoan alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,or heteroalkynylene group, as defined herein.

The term “arylalkyl,” as used herein, refers to an aryl substitutedalkyl group, wherein the terms “aryl” and “alkyl” are defined herein,and wherein the aryl group is attached to the alkyl group, which in turnis attached to the parent molecule. An exemplary arylalkyl groupincludes benzyl.

The term “aryloxy” refers to a “substituted hydroxyl” of the formula(—OR^(i)), wherein R^(i) is an optionally substituted aryl group, asdefined herein, and the oxygen moiety is directly attached to the parentmolecule.

The term “arylamino” refers to a “substituted amino” of the formula(—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted aryl group, as defined herein, and the nitrogenmoiety is directly attached to the parent molecule.

The term “arylthioxy” refers to a “substituted thiol” of the formula(—SR^(r)), wherein R^(r) is an optionally substituted aryl group, asdefined herein, and the sulfur moiety is directly attached to the parentmolecule.

The term “azido,” as used herein, refers to a group of the formula(—N₃).

The term “cyano,” as used herein, refers to a group of the formula(—CN).

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine(bromo, —Br), and iodine (iodo, —I).

The term “heteroaliphatic,” as used herein, refers to an aliphaticmoiety, as defined herein, which includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, cyclic (i.e., heterocyclic), or polycyclic hydrocarbons, whichare optionally substituted with one or more functional groups, and thatcontain one or more oxygen, sulfur, nitrogen, phosphorus, or siliconatoms, e.g., in place of carbon atoms. In certain embodiments,heteroaliphatic moieties are substituted by independent replacement ofone or more of the hydrogen atoms thereon with one or more substituents.As will be appreciated by one of ordinary skill in the art,“heteroaliphatic” is intended herein to include, but is not limited to,heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,heterocycloalkenyl, and heterocycloalkynyl moieties. Thus, the term“heteroaliphatic” includes the terms “heteroalkyl,” “heteroalkenyl,”“heteroalkynyl,” and the like. Furthermore, as used herein, the terms“heteroalkyl,” “heteroalkenyl,” “heteroalkynyl,” and the like encompassboth substituted and unsubstituted groups. In certain embodiments, asused herein, “heteroaliphatic” is used to indicate those heteroaliphaticgroups (cyclic, acyclic, substituted, unsubstituted, branched orunbranched) having 1-20 carbon atoms. Heteroaliphatic group substituentsinclude, but are not limited to, any of the substituents describedherein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano,isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino,heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, acyloxy, and the like, each of which may or may not befurther substituted).

The term “heteroalkyl,” as used herein, refers to an alkyl moiety, asdefined herein, which contain one or more oxygen, sulfur, nitrogen,phosphorus, or silicon atoms, e.g., in place of carbon atoms.

The term “heteroalkylene,” as used herein, refers to a biradical derivedfrom an heteroalkyl group, as defined herein, by removal of two hydrogenatoms. Heteroalkylene groups may be cyclic or acyclic, branched orunbranched, substituted or unsubstituted. Heteroalkylene groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “heteroalkenyl,” as used herein, refers to an alkenyl moiety,as defined herein, which contain one or more oxygen, sulfur, nitrogen,phosphorus, or silicon atoms, e.g., in place of carbon atoms.

The term “heteroalkenylene,” as used herein, refers to a biradicalderived from an heteroalkenyl group, as defined herein, by removal oftwo hydrogen atoms. Heteroalkenylene groups may be cyclic or acyclic,branched or unbranched, substituted or unsubstituted.

The term “heteroalkynyl,” as used herein, refers to an alkynyl moiety,as defined herein, which contain one or more oxygen, sulfur, nitrogen,phosphorus, or silicon atoms, e.g., in place of carbon atoms.

The term “heteroalkynylene,” as used herein, refers to a biradicalderived from an heteroalkynyl group, as defined herein, by removal oftwo hydrogen atoms. Heteroalkynylene groups may be cyclic or acyclic,branched or unbranched, substituted or unsubstituted.

The term “heteroalkylamino” refers to a “substituted amino” of theformula (—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted heteroalkyl group, as defined herein, and thenitrogen moiety is directly attached to the parent molecule.

The term “heteroalkyloxy” refers to a “substituted hydroxyl” of theformula (—OR^(i)), wherein R^(i) is an optionally substitutedheteroalkyl group, as defined herein, and the oxygen moiety is directlyattached to the parent molecule.

The term “heteroalkylthioxy” refers to a “substituted thiol” of theformula (—SR), wherein R^(r) is an optionally substituted heteroalkylgroup, as defined herein, and the sulfur moiety is directly attached tothe parent molecule.

The term “heterocyclic,” “heterocycles,” or “heterocyclyl,” as usedherein, refers to a cyclic heteroaliphatic group. A heterocyclic grouprefers to a non-aromatic, partially unsaturated or fully saturated, 3-to 10-membered ring system, which includes single rings of 3 to 8 atomsin size, and bi- and tri-cyclic ring systems which may include aromaticfive- or six-membered aryl or heteroaryl groups fused to a non-aromaticring. These heterocyclic rings include those having from one to threeheteroatoms independently selected from oxygen, sulfur, and nitrogen, inwhich the nitrogen and sulfur heteroatoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. In certainembodiments, the term heterocyclic refers to a non-aromatic 5-, 6-, or7-membered ring or polycyclic group wherein at least one ring atom is aheteroatom selected from O, S, and N (wherein the nitrogen and sulfurheteroatoms may be optionally oxidized), and the remaining ring atomsare carbon, the radical being joined to the rest of the molecule via anyof the ring atoms. Heterocycyl groups include, but are not limited to, abi- or tri-cyclic group, comprising fused five, six, or seven-memberedrings having between one and three heteroatoms independently selectedfrom the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ringhas 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds,and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen andsulfur heteroatoms may be optionally oxidized, (iii) the nitrogenheteroatom may optionally be quaternized, and (iv) any of the aboveheterocyclic rings may be fused to an aryl or heteroaryl ring. Exemplaryheterocycles include azacyclopropanyl, azacyclobutanyl,1,3-diazatidinyl, piperidinyl, piperazinyl, azocanyl, thiaranyl,thietanyl, tetrahydrothiophenyl, dithiolanyl, thiacyclohexanyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropuranyl, dioxanyl,oxathiolanyl, morpholinyl, thioxanyl, tetrahydronaphthyl, and the like,which may bear one or more substituents. Substituents include, but arenot limited to, any of the substituents described herein, that result inthe formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl,alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl,sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “heteroaryl,” as used herein, refer to stable aromatic mono- orpolycyclic ring system having 3-20 ring atoms, of which one ring atom isselected from S, O, and N; zero, one, or two ring atoms are additionalheteroatoms independently selected from S, O, and N; and the remainingring atoms are carbon, the radical being joined to the rest of themolecule via any of the ring atoms. Exemplary heteroaryls include, butare not limited to pyrrolyl, pyrazolyl, imidazolyl, pyridinyl,pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl,pyyrolizinyl, indolyl, quinolinyl, isoquinolinyl, benzoimidazolyl,indazolyl, quinolinyl, isoquinolinyl, quinolizinyl, cinnolinyl,quinazolynyl, phthalazinyl, naphthridinyl, quinoxalinyl, thiophenyl,thianaphthenyl, furanyl, benzofuranyl, benzothiazolyl, thiazolynyl,isothiazolyl, thiadiazolynyl, oxazolyl, isoxazolyl, oxadiaziolyl,oxadiaziolyl, and the like, which may bear one or more substituents.Heteroaryl substituents include, but are not limited to, any of thesubstituents described herein, that result in the formation of a stablemoiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic,heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

The term “heteroarylene,” as used herein, refers to a biradical derivedfrom an heteroaryl group, as defined herein, by removal of two hydrogenatoms. Heteroarylene groups may be substituted or unsubstituted.Additionally, heteroarylene groups may be incorporated as a linker groupinto an alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, or heteroalkynylene group, as defined herein.Heteroarylene group substituents include, but are not limited to, any ofthe substituents described herein, that result in the formation of astable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

The term “heteroarylamino” refers to a “substituted amino” of the(—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted heteroaryl group, as defined herein, and thenitrogen moiety is directly attached to the parent molecule.

The term “heteroaryloxy” refers to a “substituted hydroxyl” of theformula (—OR^(i)), wherein R^(i) is an optionally substituted heteroarylgroup, as defined herein, and the oxygen moiety is directly attached tothe parent molecule.

The term “heteroarylthioxy” refers to a “substituted thiol” of theformula (—SR), wherein R^(r) is an optionally substituted heteroarylgroup, as defined herein, and the sulfur moiety is directly attached tothe parent molecule.

The term “hydroxy,” or “hydroxyl,” as used herein, refers to a group ofthe formula (—OH). A “substituted hydroxyl” refers to a group of theformula (—OR^(i)), wherein R^(i) can be any substituent which results ina stable moiety (e.g., a suitable hydroxyl protecting group; aliphatic,alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, nitro, alkylaryl, arylalkyl, and the like, each ofwhich may or may not be further substituted).

The term “imino,” as used herein, refers to a group of the formula(═NR^(r)), wherein R^(r) corresponds to hydrogen or any substituent asdescribed herein, that results in the formation of a stable moiety (forexample, a suitable amino protecting group; aliphatic, alkyl, alkenyl,alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, amino,hydroxyl, alkylaryl, arylalkyl, and the like, each of which may or maynot be further substituted).

The term “isocyano,” as used herein, refers to a group of the formula(—NC).

The term “nitro,” as used herein, refers to a group of the formula(—NO₂).

The term “oxo,” as used herein, refers to a group of the formula (═O).

As used herein, the term “resin” refers to a resin useful for solidphase synthesis. Solid phase synthesis is a well-known synthetictechnique; see generally, Atherton, E., Sheppard, R. C. Solid PhasePeptide Synthesis: A Practical Approach, IRL Press, Oxford, England,1989, and Stewart J. M., Young, J. D. Solid Phase Peptide Synthesis, 2ndedition, Pierce Chemical Company, Rockford, 1984, the entire contents ofeach of which are hereby incorporated herein by reference. Exemplaryresins which may be employed by the present invention include, but arenot limited to:

(1) alkenyl resins (e.g., REM resin, vinyl sulfone polymer-bound resin,vinyl-polystyrene resin);

(2) amine functionalized resins (e.g., amidine resin,N-(4-Benzyloxybenzyl)hydroxylamine polymer bound,(aminomethyl)polystyrene, polymer bound (R)-(+)-a-methylbenzylamine,2-Chlorotrityl Knorr resin, 2-N-Fmoc-Amino-dibenzocyclohepta-1,4-diene,polymer-bound resin,4-[4-(1-Fmoc-aminoethyl)-2-methoxy-5-nitrophenoxy]butyramidomethyl-polystyreneresin, 4-Benzyloxybenzylamine, polymer-bound,4-Carboxybenzenesulfonamide, polymer-bound,Bis(tert-butoxycarbonyl)thiopseudourea, polymer-bound,Dimethylaminomethyl-polystyrene, Fmoc-3-amino-3-(2-nitrophenyl)propionicacid, polymer-bound, N-Methyl aminomethylated polystyrene, PAL resin,Sieber amide resin, tert-Butyl N-(2-mercaptoethyl)carbamate,polymer-bound, Triphenylchloromethane-4-carboxamide polymer bound);

(3) benzhydrylamine (BHA) resins (e.g., 2-Chlorobenzhydryl chloride,polymer-bound, HMPB-benzhydrylamine polymer bound, 4-Methylbenzhydrol,polymer-bound, Benzhydryl chloride, polymer-bound, Benzhydrylaminepolymer-bound);

(4) Br-functionalized resins (e.g., 4-(Benzyloxy)benzyl bromide polymerbound, 4-Bromopolystyrene, Brominated PPOA resin, Brominated Wang resin,Bromoacetal, polymer-bound, Bromopolystyrene, HypoGel® 200 Br,Polystyrene A-Br for peptide synthesis, Selenium bromide, polymer-bound,TentaGel HL-Br, TentaGel MB-Br, TentaGel S-Br, TentaGel S-Br);

(5) Chloromethyl resins (e.g., 5-[4-(Chloromethyl)phenyl]pentyl]styrene,polymer-bound, 4-(Benzyloxy)benzyl chloride polymer bound,4-Methoxybenzhydryl chloride, polymer-bound);

(6) CHO-functionalized resins (e.g.,(4-Formyl-3-methoxyphenoxymethyl)polystyrene,(4-Formyl-3-methoxyphenoxymethyl)polystyrene, 3-Benzyloxybenzaldehyde,polymer-bound, 4-Benzyloxy-2,6-dimethoxybenzaldehyde, polymer-bound,Formylpolystyrene, HypoGel® 200 CHO, Indole resin, PolystyreneA-CH(OEt)₂, TentaGel HL-CH(OEt)₂);

(7) Cl-functionalized resins (e.g., Benzoyl chloride polymer bound,(chloromethyl)polystyrene, Merrifield's resin);

(8) CO₂Hfunctionalized resins (e.g., Carboxyethylpolystryrene, HypoGel®200 COOH, Polystyrene AM-COOH, TentaGel HL-COOH, TentaGel MB—COOH,TentaGel S—COOH);

(9) Hypo-Gel resins (e.g., HypoGel® 200 FMP, HypoGel® 200 PHB, HypoGel®200 Trt-OH, HypoGel® 200 HMB);

(10) I-functionalized resins (e.g., 4-Iodophenol, polymer-bound,Iodopolystyrene); Janda-Jels™ (JandaJel^(a)-Rink amide, JandaJel-NH₂,JandaJel-Cl, JandaJel-4-Mercaptophenol, JandaJel-OH,JandaJel-1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide,JandaJel-1,3,4,6,7,8-hexahydro-2H-pyrimido-[1,2-a] pyrimidine,JandaJel-morpholine, JandaJel-polypyridine, JandaJel-Triphenylphosphine,JandaJel-Wang);

(11) MBHA resins (3[4′-(Hydroxymethyl)phenoxy] propionicacid-4-methylbenzhydrylamine resin, 4-(Hydroxymethyl)phenoxyacetic acidpolymer-bound to MBHA resin, HMBA-4-methylbenzhydrylamine polymer bound,4-Methylbenzhydrylamine hydrochloride polymer bound Capacity (amine));

(12) NH₂ functionalized resins ((Aminomethyl)polystyrene,(Aminomethyl)polystyrene, HypoGel® 200 NH₂, Polystyrene AM-NH₂,Polystyrene Microspheres 2-aminoethylated, Polystyrol Microspheres2-bromoethylated, Polystyrol Microspheres 2-hydroxyethylated, TentaGelHL-NH₂, Tentagel M Br, Tentagel M NH₂, Tentagel M OH, TentaGel MB-NH₂,TentaGel S—NH₂, TentaGel S—NH₂);

(13) OH-functionalized resins (e.g., 4-hydroxymethylbenzoic acid,polymer-bound, Hydroxymethyl Resins, OH-functionalized Wang Resins);

(14) oxime resins (e.g., 4-Chlorobenzophenone oxime polymer bound,Benzophenone oxime polymer bound, 4-Methoxybenzophenone oxime polymerbound);

(15) PEG resins (e.g., ethylene glycol polymer bound);

(16) Boc-/Blz peptide synthesis resins (e.g.,Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]-Cys(Acm)-b-Ala-O-PAM resin,Boc-Lys(Fmoc)-Lys[Boc-Lys(Fmoc)]-b-Ala-O-Pam resin,Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]-Lys{Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]}-b-Ala-O-PAMresin,Boc-Lys(Fmoc)-Lys[Boc-Lys(Fmoc)]-Lys{Boc-Lys(Fmoc)-Lys[Boc-Lys(Fmoc)]}-b-Ala-O-PAMresin,Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]-Lys{Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]}-Cys(Acm)-b-Ala-O-PAMresin, Preloaded PAM resins);

(17) Fmoc-/t-Bu peptide synthesis resins (e.g.,Fmoc-Lys(Fmoc)-Lys[Fmoc-Lys(Fmoc)]-b-Ala-O-Wang resin,Fmoc-Lys(Fmoc)-Lys[Fmoc-Lys(Fmoc)]-Lys{Fmoc-Lys(Fmoc)-Lys[Fmoc-Lys(Fmoc)]}-b-Ala-O-Wangresin, Preloaded TentaGel® S Trityl Resins, Preloaded TentaGel® Resins,Preloaded Trityl Resins, Preloaded Wang Resins, Trityl Resins Preloadedwith Amino Alcohols);

(19) thiol-functionalized resins (e.g., HypoGel® 200 S-Trt, PolystyreneAM-S-Trityl, TentaGel HL-S-Trityl, TentaGel MB-S-Trityl, TentaGelS—S-Trityl); and

(20) Wang resins (e.g., Fmoc-Ala-Wang resin, Fmoc-Arg(Pbf)-Wang resin,Fmoc-Arg(Pmc)-Wang resin, Fmoc-Asn(Trt)-Wang resin, Fmoc-Asp(OtBu)-Wangresin, Fmoc-Cys(Acm)-Wang resin, Fmoc-Cys(StBu)-Wang resin,Fmoc-Cys(Trt) Wang resin, Fmoc-Gln(Trt)-Wang resin, Fmoc-Glu(OtBu)-Wangresin, Fmoc-Gly-Wang resin, Fmoc-His(Trt)-Wang resin, Fmoc-Ile-Wangresin, Fmoc-Leu-Wang resin, Fmoc-Lys(Boc)-Wang resin, Fmoc-Met-Wangresin, Fmoc-D-Met-Wang resin, Fmoc-Phe-Wang resin, Fmoc-Pro-Wang resin,Fmoc-Ser(tBu)-Wang resin, Fmoc-Ser(Trt)-Wang resin, Fmoc-Thr(tBu)-Wangresin, Fmoc-Trp(Boc) Wang resin, Fmoc-Trp-Wang resin, Fmoc-Tyr(tBu)-Wangresin, Fmoc-Val-Wang resin).

The term “stable moiety,” as used herein, preferably refers to a moietywhich possess stability sufficient to allow manufacture, and whichmaintains its integrity for a sufficient period of time to be useful forthe purposes detailed herein.

A “suitable amino-protecting group,” as used herein, is well known inthe art and include those described in detail in Protecting Groups inOrganic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, JohnWiley & Sons, 1999, the entirety of which is incorporated herein byreference. Suitable amino-protecting groups include methyl carbamate,ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N′dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

A “suitable carboxylic acid protecting group” or “protected carboxylicacid,” as used herein, are well known in the art and include thosedescribed in detail in Greene (1999). Examples of suitably protectedcarboxylic acids further include, but are not limited to, silyl-,alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids.Examples of suitable silyl groups include trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and thelike. Examples of suitable alkyl groups include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl,tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl.Examples of suitable aryl groups include optionally substituted phenyl,biphenyl, or naphthyl. Examples of suitable arylalkyl groups includeoptionally substituted benzyl (e.g., p-methoxybenzyl (MPM),3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

A “suitable hydroxyl protecting group,” as used herein, is well known inthe art and include those described in detail in Protecting Groups inOrganic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, JohnWiley & Sons, 1999, the entirety of which is incorporated herein byreference. Suitable hydroxyl protecting groups include methyl,methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

A “suitable thiol protecting group,” as used herein, are well known inthe art and include those described in detail in Protecting Groups inOrganic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, JohnWiley & Sons, 1999, the entirety of which is incorporated herein byreference. Examples of suitably protected thiol groups further include,but are not limited to, thioesters, carbonates, sulfonates allylthioethers, thioethers, silyl thioethers, alkyl thioethers, arylalkylthioethers, and alkyloxyalkyl thioethers. Examples of suitable estergroups include formates, acetates, proprionates, pentanoates,crotonates, and benzoates. Specific examples of suitable ester groupsinclude formate, benzoyl formate, chloroacetate, trifluoroacetate,methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate,pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benylbenzoate, 2,4,6-trimethylbenzoate. Examples of suitablecarbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl,2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, andp-nitrobenzyl carbonate. Examples of suitable silyl groups includetrimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilylethers. Examples of suitable alkyl groups include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether,or derivatives thereof. Examples of suitable arylalkyl groups includebenzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and4-picolyl ethers.

The term “thio” or “thiol,” as used herein, refers to a group of theformula (—SH). A “substituted thiol” refers to a group of the formula(—SR), wherein R can be any substituent that results in the formation ofa stable moiety (e.g., a suitable thiol protecting group; aliphatic,alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, sulfinyl, sulfonyl, cyano, nitro, alkylaryl,arylalkyl, and the like, each of which may or may not be furthersubstituted).

The term “thiooxo,” as used herein, refers to a group of the formula(═S).

As used herein, a “pharmaceutically acceptable form thereof” includesany pharmaceutically acceptable salts, prodrugs, tautomers, enantiomers,diastereomers, stereoisomers, isomers, and/or polymorphs of a compoundof the present invention, as defined below and herein.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.,describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

As used herein, the term “prodrug” refers to a derivative of a parentcompound that requires transformation within the body in order torelease the parent compound. In certain cases, a prodrug has improvedphysical and/or delivery properties over the parent compound. Prodrugsare typically designed to enhance pharmaceutically and/orpharmacokinetically based properties associated with the parentcompound. The advantage of a prodrug can lie in its physical properties,such as enhanced water solubility for parenteral administration atphysiological pH compared to the parent compound, or it enhancesabsorption from the digestive tract, or it may enhance drug stabilityfor long-term storage. In recent years several types of bioreversiblederivatives have been exploited for utilization in designing prodrugs.Using esters as a prodrug type for compounds containing a carboxyl orhydroxyl functionality is known in the art as described, for example, in“The Organic Chemistry of Drug Design and Drug Interaction” RichardSilverman, published by Academic Press (1992).

As used herein, the term “tautomer” includes two or moreinterconvertable compounds resulting from at least one formal migrationof a hydrogen atom and at least one change in valency (e.g., a singlebond to a double bond, a triple bond to a double bond, or vice versa).The exact ratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Tautomerizations (i.e., the reactionproviding a tautomeric pair) may catalyzed by acid or base. Exemplarytautomerizations include keto-to-enol; amide-to-imide; lactam-to-lactim;enamine-to-imine; and enamine-to-(a different) enamine tautomerizations.

As used herein, the term “isomers” includes any and all geometricisomers and stereoisomers. For example, “isomers” include cis- andtrans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers,(D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixturesthereof, as falling within the scope of the invention. For instance, anisomer/enantiomer may, in some embodiments, be provided substantiallyfree of the corresponding enantiomer, and may also be referred to as“optically enriched.” “Optically-enriched,” as used herein, means thatthe compound is made up of a significantly greater proportion of oneenantiomer. In certain embodiments the compound of the present inventionis made up of at least about 90% by weight of a preferred enantiomer. Inother embodiments the compound is made up of at least about 95%, 98%, or99% by weight of a preferred enantiomer. Preferred enantiomers may beisolated from racemic mixtures by any method known to those skilled inthe art, including chiral high pressure liquid chromatography (HPLC) andthe formation and crystallization of chiral salts or prepared byasymmetric syntheses. See, for example, Jacques, et al., Enantiomers,Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen,S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistryof Carbon Compounds (McGraw-Hill, N Y, 1962); Wilen, S. H. Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972).

The term “amino acid” refers to a molecule containing both an aminogroup and a carboxyl group. Amino acids include alpha-amino acids andbeta-amino acids, the structures of which are depicted below. In certainembodiments, an amino acid is an alpha amino acid.

Suitable amino acids include, without limitation, natural alpha-aminoacids such as D- and L-isomers of the 20 common naturally occurringalpha-amino acids found in peptides and proteins (e.g., A, R, N, C, D,Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as depicted in Table 1below), unnatural alpha-amino acids (as depicted in Tables 2 and 3below), natural beta-amino acids (e.g., beta-alanine), and unnaturalbeta-amino acids.

Amino acids used in the construction of peptides of the presentinvention may be prepared by organic synthesis, or obtained by otherroutes, such as, for example, degradation of or isolation from a naturalsource. In certain embodiments of the present invention, the formula—[X_(AA)]— corresponds to the natural and/or unnatural amino acidshaving the following formulae:

wherein R and R′ correspond a suitable amino acid side chain, as definedherein, and R^(a) is as defined herein.

TABLE 1 Exemplary natural alpha- Suitable amino acid side chains aminoacids R R′ L-Alanine (A) —CH₃ —H L-Arginine (R) —CH₂CH₂CH₂—NHC(═NH)NH₂—H L-Asparagine (N) —CH₂C(═O)NH₂ —H L-Aspartic acid (D) —CH₂CO₂H —HL-Cysteine (C) —CH₂SH —H L-Glutamic acid (E) —CH₂CH₂CO₂H —H L-Glutamine(Q) —CH₂CH₂C(═O)NH₂ —H Glycine (G) —H —H L-Histidine (H)—CH₂-2-(1H-imidazole) —H L-Isoleucine (I) -sec-butyl —H L-Leucine (L)-iso-butyl —H L-Lysine (K) —CH₂CH₂CH₂CH₂NH₂ —H L-Methionine (M)—CH₂CH₂SCH₃ —H L-Phenylalanine (F) —CH₂Ph —H L-Proline (P)-2-(pyrrolidine) —H L-Serine (S) —CH₂OH —H L-Threonine (T)—CH₂CH(OH)(CH₃) —H L-Tryptophan (W) —CH₂-3-(1H-indole) —H L-Tyrosine (Y)—CH₂-(p-hydroxyphenyl) —H L-Valine (V) -isopropyl —H

TABLE 2 Exemplary unnatural Suitable amino acid side chains alpha-aminoacids R R′ D-Alanine —H —CH₃ D-Arginine —H —CH₂CH₂CH₂—NHC(═NH)NH₂D-Asparagine —H —CH₂C(═O)NH₂ D-Aspartic acid —H —CH₂CO₂H D-Cysteine —H—CH₂SH D-Glutamic acid —H —CH₂CH₂CO₂H D-Glutamine —H —CH₂CH₂C(═O)NH₂D-Histidine —H —CH₂-2-(1H-imidazole) D-Isoleucine —H -sec-butylD-Leucine —H -iso-butyl D-Lysine —H —CH₂CH₂CH₂CH₂NH₂ D-Methionine —H—CH₂CH₂SCH₃ D-Phenylalanine —H —CH₂Ph D-Proline —H -2-(pyrrolidine)D-Serine —H —CH₂OH D-Threonine —H —CH₂CH(OH)(CH₃) D-Tryptophan —H—CH₂-3-(1H-indole) D-Tyrosine —H —CH₂-(p-hydroxyphenyl) D-Valine —H-isopropyl Di-vinyl —CH═CH₂ —CH═CH₂ Exemplary unnatural alpha-aminoacids R and R′ are equal to: α-methyl-Alanine —CH₃ —CH₃ (Aib)α-methyl-Arginine —CH₃ —CH₂CH₂CH₂—NHC(═NH)NH₂ α-methyl-Asparagine —CH₃—CH₂C(═O)NH₂ α-methyl-Aspartic —CH₃ —CH₂CO₂H acid α-methyl-Cysteine —CH₃—CH₂SH α-methyl-Glutamic —CH₃ —CH₂CH₂CO₂H acid α-methyl-Glutamine —CH₃—CH₂CH₂C(═O)NH₂ α-methyl-Histidine —CH₃ —CH₂-2-(1H-imidazole)α-methyl-Isoleucine —CH₃ -sec-butyl α-methyl-Leucine —CH₃ -iso-butylα-methyl-Lysine —CH₃ —CH₂CH₂CH₂CH₂NH₂ α-methyl-Methionine —CH₃—CH₂CH₂SCH₃ α-methyl-Phenylal- —CH₃ —CH₂Ph anine α-methyl-Proline —CH₃-2-(pyrrolidine) α-methyl-Serine —CH₃ —CH₂OH α-methyl-Threonine —CH₃—CH₂CH(OH)(CH₃) α-methyl-Tryptophan —CH₃ —CH₂-3-(1H-indole)α-methyl-Tyrosine —CH₃ —CH₂-(p-hydroxyphenyl) α-methyl-Valine —CH₃-isopropyl Di-vinyl —CH═CH₂ —CH═CH₂ Norleucine —H —CH₂CH₂CH₂CH₃

TABLE 3 Exemplary unnatural alpha-amino Suitable amino acid side chainsR and R′ acids is equal to hydrogen or —CH₃, and: Terminally unsaturatedalpha-amino —(CH₂)_(g)—S—(CH₂)_(g)CH═CH₂, acids and bis alpha-aminoacids (e.g., —(CH₂)_(g)—O—(CH₂)_(g)CH═CH₂, modified cysteine, modifiedlysine, —(CH₂)_(g)—NH—(CH₂)_(g)CH═CH₂, modified tryptophan, modifiedserine, —(CH₂)_(g)—(C═O)—S—(CH₂)_(g)CH═CH₂, modified threonine, modifiedproline, —(CH₂)_(g)—(C═O)—O—(CH₂)_(g)CH═CH₂, modified histidine,modified alanine, —(CH₂)_(g)—(C═O)—NH—(CH₂)_(g)CH═CH₂, and the like).—CH₂CH₂CH₂CH₂—NH—(CH₂)_(g)CH═CH₂, —(C₆H₅)—p-O—(CH₂)_(g)CH═CH_(2,)—CH(CH₃)—O—(CH₂)_(g)CH═CH₂, —CH₂CH(—O—CH═CH₂)(CH₃),-histidine—N((CH₂)_(g)CH═CH₂), -tryptophan—N((CH₂)_(g)CH═CH₂), and—(CH₂)_(g+1)(CH═CH₂), wherein: each instance of g is, dependently, 0 to10, inclusive. Exemplary unnatural alpha-amino acids

R₅

R₈

S₅

S₈

B₅

There are many known unnatural amino acids any of which may be includedin the peptides of the present invention. See, for example, S. Hunt, TheNon-Protein Amino Acids: In Chemistry and Biochemistry of the AminoAcids, edited by G. C. Barrett, Chapman and Hall, 1985. Some examples ofunnatural amino acids are 4-hydroxyproline, desmosine,gamma-aminobutyric acid, beta-cyanoalanine, norvaline,4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine,1-amino-cyclopropanecarboxylic acid,1-amino-2-phenyl-cyclopropanecarboxylic acid,1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid,3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioicacid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta-and para-substituted phenylalanines (e.g., substituted with —C(═O)C₆H₅;—CF₃; —CN; -halo; —NO₂; —CH₃), disubstituted phenylalanines, substitutedtyrosines (e.g., further substituted with —C(═O)C₆H₅; —CF₃; —CN; -halo;—NO₂; —CH₃), and statine. Additionally, the amino acids suitable for usein the present invention may be derivatized to include amino acidresidues that are hydroxylated, phosphorylated, sulfonated, acylated,lipidated, and glycosylated, to name a few.

The term “amino acid side chain” refers to a group attached to thealpha- or beta-carbon of an amino acid. A “suitable amino acid sidechain” includes, but is not limited to, any of the suitable amino acidside chains as defined above, and as provided in Tables 1 to 3.

For example, suitable amino acid side chains include methyl (as thealpha-amino acid side chain for alanine is methyl),4-hydroxyphenylmethyl (as the alpha-amino acid side chain for tyrosineis 4-hydroxyphenylmethyl) and thiomethyl (as the alpha-amino acid sidechain for cysteine is thiomethyl), etc. A “terminally unsaturated aminoacid side chain” refers to an amino acid side chain bearing a terminalunsaturated moiety, such as a substituted or unsubstituted, double bond(e.g., olefinic) or a triple bond (e.g., acetylenic), that participatesin a crosslinking reaction with other terminal unsaturated moieties inthe polypeptide chain. In certain embodiments, a “terminally unsaturatedamino acid side chain” is a terminal olefinic amino acid side chain. Incertain embodiments, a “terminally unsaturated amino acid side chain” isa terminal acetylenic amino acid side chain. In certain embodiments, theterminal moiety of a “terminally unsaturated amino acid side chain” isnot further substituted. Terminally unsaturated amino acid side chainsinclude, but are not limited to, side chains as depicted in Table 3.

A “peptide,” “protein,” “polypeptide,” or “peptidic” comprises a polymerof amino acid residues linked together by peptide (amide) bonds. Theterm(s), as used herein, refers to proteins, polypeptides, and peptideof any size, structure, or function. Typically, a peptide or polypeptidewill be at least three amino acids long. A peptide or polypeptide mayrefer to an individual protein or a collection of proteins. Inventiveproteins preferably contain only natural amino acids, althoughnon-natural amino acids (i.e., compounds that do not occur in nature butthat can be incorporated into a polypeptide chain) and/or amino acidanalogs as are known in the art may alternatively be employed. Also, oneor more of the amino acids in a peptide or polypeptide may be modified,for example, by the addition of a chemical entity such as a carbohydrategroup, a hydroxyl group, a phosphate group, a farnesyl group, anisofarnesyl group, a fatty acid group, a linker for conjugation,functionalization, or other modification, etc. A peptide or polypeptidemay also be a single molecule or may be a multi-molecular complex. Apeptide or polypeptide may be just a fragment of a naturally occurringprotein or peptide. A peptide or polypeptide may be naturally occurring,recombinant, or synthetic, or any combination thereof.

The following definitions are more general terms used throughout thepresent application:

The term “subject,” as used herein, refers to any animal. In certainembodiments, the subject is a mammal. In certain embodiments, the term“subject”, as used herein, refers to a human (e.g., a man, a woman, or achild) of either sex at any stage of development.

The terms “administer,” “administering,” or “administration,” as usedherein refers to implanting, applying, absorbing, ingesting, injecting,or inhaling, the inventive polypeptide or compound.

The terms “treat” or “treating,” as used herein, refers to partially orcompletely alleviating, inhibiting, ameliorating, and/or relieving thedisease or condition from which the subject is suffering.

The terms “effective amount” and “therapeutically effective amount,” asused herein, refer to the amount or concentration of a biologicallyactive agent conjugated to an inventive polypeptide of the presentlyclaimed invention, or amount or concentration of an inventivepolypeptide, that, when administered to a subject, is effective to atleast partially treat a condition from which the subject is suffering.

As used herein, when two entities are “associated with” one another theyare linked by a direct or indirect covalent or non-covalent interaction.In certain embodiments, the association is covalent and the entities are“conjugated.” In other embodiments, the association is non-covalent.Non-covalent interactions include hydrogen bonding, van der Waalsinteractions, hydrophobic interactions, magnetic interactions,electrostatic interactions, etc. An indirect covalent interaction iswhen two entities are covalently associated through a linker.

As used herein, a “label” refers to a moiety that has at least oneelement, isotope, or functional group incorporated into the moiety whichenables detection of the inventive polypeptide to which the label isattached. Labels can be directly attached (i.e., via a bond) or can beattached by a tether (such as, for example, a cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedalkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkynylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted heteroalkenylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkynylene; substituted or unsubstituted arylene; substituted orunsubstituted heteroarylene; or substituted or unsubstituted acylene, orany combination thereof, which can make up a tether). It will beappreciated that the label may be attached to or incorporated into theinventive polypeptide at any position.

In general, a label can fall into any one (or more) of five classes: a)a label which contains isotopic moieties, which may be radioactive orheavy isotopes, including, but not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N,³¹P, ³²P, ³⁵S, ⁶⁷Ga, ^(99m)Tc (Tc-99m), ¹¹¹In, ¹²³I, ¹²⁵I, ¹⁶⁹Yb, and¹⁸⁶Re; b) a label which contains an immune moiety, which may beantibodies or antigens, which may be bound to enzymes (e.g., such ashorseradish peroxidase); c) a label which is a colored, luminescent,phosphorescent, or fluorescent moieties (e.g., such as the fluorescentlabel FITC); d) a label which has one or more photo affinity moieties;and e) a label which has a ligand moiety with one or more known bindingpartners (such as biotin-streptavidin, FK506-FKBP, etc.).

In certain embodiments, a label comprises a radioactive isotope,preferably an isotope which emits detectable particles, such as Rparticles.

In certain embodiments, the label comprises a fluorescent moiety. Incertain embodiments, the label is the fluorescent label FITC. In certainembodiments, the label comprises a ligand moiety with one or more knownbinding partners. In certain embodiments, the label comprises the ligandmoiety biotin.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the endogenous β-catenin degradation pathway as adaptedfrom Barker and Clevers, NRDD, 5, 998-1014 (2006), incorporated hereinby reference.

FIG. 2 shows the loss of endogenous β-catenin degradation in humancancers.

FIG. 3 depicts restoration of β-catenin destruction using a bifunctionalstapled peptide.

FIG. 4 shows surface exposed lysines on the β-catenin Arm repeat domainthat are putative sites for ubiquitination.

FIG. 5 shows an example of a bifunctional stapled peptide based on Bcl9and p53 that can bring hDM2 in close proximity to β-catenin to effectubiquitination.

FIG. 6 shows an example of a bifunctional stapled peptide based on Tcf4and p53 that can bring hDM2 in close proximity to β-catenin to effectubiquitination.

FIG. 7 depicts examples of bifunctional stapled peptides Tcf4-SAH p53-8,SAH p53-8-Tcf4, Bcl-9-SAH p53-8, SAH p53-8-Bcl-9 with differenceorientations.

FIG. 8 depicts example sequences of bifunctional stapled peptides SAHp53-8-Bcl-9 (SEQ ID NO: 1-3) and Bcl-9-SAH p53-8 (SEQ ID NO: 4-6) withAhx linker.

FIG. 9 depicts example sequences of bifunctional stapled peptides SAHp53-8-Bcl-9 (SEQ ID NO: 7-9) and Bcl-9-SAH p53-8 (SEQ ID NO: 10-12) withPEG linker.

FIG. 10 depicts example sequences of bifunctional stapled peptides SAHp53-8-Tcf4 (SEQ ID NO: 13, 14) and Tcf4-SAH p53-8 (SEQ ID NO: 15, 16)with Ahx linker.

FIG. 11 depicts example sequences of bifunctional stapled peptides SAHp53-8-Tcf4 (SEQ ID NO: 17, 18) and Tcf4-SAH p53-8 (SEQ ID NO: 19, 20)with PEG linker.

FIG. 12 depicts examples using cross-linkers to join the two peptidedomains (targeting domain and effector domain).

FIG. 13 depicts examples of different types of spacers between NHS andmaleimide.

FIG. 14 depicts examples of segment cross-linking in differentorientations.

FIG. 15 depicts an example of a screening procedure for high affinitybinding of synthetic libraries of stapled peptides to targets.

FIG. 16 depicts an example of a screening procedure for high affinitybinding of synthetic libraries of stapled peptides to targets. Thescreening procedure includes the detection of a modification of a secondprotein as a criterion for selection.

FIG. 17 depicts a diagram showing degradation through targetedubiquitination.

FIG. 18 depicts a diagram showing target gene repression throughrecruitment of co-repressors.

FIG. 19 depicts a diagram showing transcription factor inhibition bytargeted nuclear export with Nuclear Export Sequence (NES)-containingbi-functional peptides.

FIG. 20 depicts a diagram showing transcription factor activation bytargeted nuclear import with nuclear localization sequence(NLS)-containing bifunctional peptides.

FIG. 21 depicts a diagram showing synthetic transcription factoractivation by recruitment of co-activator proteins.

FIG. 22 depicts a diagram showing general transcription factorpost-translational modification by tethered effector domains.

FIG. 23 depicts a diagram showing design and synthesis of bifunctionalstapled peptides and attachment strategies.

FIGS. 24A-24B depicts molecular models. FIG. 24A is a model showingSin3/Mad1 interaction (Geuzennec et al., J. Biol. Chem., 2004. 279,25823-9; incorporated herein by reference). FIG. 24B is a model showingKIX/c-Myb and KIX-MLL interaction (KIX:c-Myb: Zor et al., JMB, 2004,337, 521-34, incorporated herein by reference; KIX:MLL: Guzman et al.,JMB, 2005, 355, 1005-13, incorporated herein by reference).

FIGS. 25A-25C show the sequences and biological activity of exemplaryrepressive domains. FIG. 25A shows sequences of SID peptide and stapledversions thereof. Asterisks indicate the incorporation of non-naturalamino acids for peptide stapling.

FIG. 25B shows sample fluorescent polarization experiment data for SID2and SID5 as compared to wild type SID used to determine dissociationconstants (K_(D)). FIG. 25C shows confocal microscopy of Hela cellstreated with FITC-conjugated SID-series peptides. SID2 and SID5 revealrobust cellular penetration.

FIGS. 26A-26C show the sequences and biological activity of exemplaryactivation domains. FIG. 26A shows sequences of MLL and cMyb peptidesand stapled versions thereof. Asterisks indicate the incorporation ofnon-natural amino acids for peptide stapling. NT: not tested. FIG. 26Bshows sample fluorescent polarization experiment data for MLL1-2 used todetermine dissociation constants (K_(D)). FIG. 26C shows confocalmicroscopy of U2OS cells treated with FITC-conjugated MLL-seriespeptides. MLL1-2 reveals robust cellular penetration.

FIG. 27 depicts a diagram showing examples of design and synthesis ofbifunctional stapled peptides and attachment strategies.

FIG. 28 depicts a diagram showing the synthesis of a stapled peptidecontaining a maleimide reactive group.

FIG. 29 depicts a diagram showing the synthesis of a stapled peptidecontaining a thiol reactive group.

FIG. 30 depicts a diagram showing a conjugated bifunctional stapledpeptide associated via reaction of a thiol-containing stapled peptideand a maleimide-containing stapled peptide.

FIG. 31 depicts mass spectrum for a thiol-containing stapled peptide(upper panel), a maleimide-containing stapled peptide (middle panel),and the resulting conjugated bifunctional stapled peptide (lower panel).

FIG. 32 depicts mass spectrum of an HPLC-purified conjugatedbifunctional stapled peptide.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention stems from the recognition of a new use forstapled or stitched peptides. Given the stability of such peptides theymay be used as agents for recruiting proteins or other biomolecules to aparticular protein, nucleic acid, other biomolecule, cell, or organelleor other cellular entities. The invention thus relates to bifunctionalstapled or stitched peptides that can tether, or bring together cellularentities. One domain of the bifunctional peptide acts as a targetingmoiety that binds to a target; the other domain acts as an effectordomain to recruit a protein, protein complex, or other biomolecule tothe target. In essence, the bifunctional peptide works to bring twoproteins or other biomolecules in proximity to one another. Thetargeting domain, the effector domain, or both domains may be stapled orstitched to stabilize the conformation of the peptide.

In certain embodiments, bifunctional stapled or stitched peptides of theinvention can be used to tether any two biomolecules (such aspolypeptides) together. A polypeptide can be, for example, a singlepolypeptide, such as a protein, or can be a complex comprising two ormore polypeptides that associate with each other, such as a proteincomplex.

To tether, as used herein, means to bring into close proximity cellularentities (e.g., proteins, nucleic acids, membranes, organelles, etc.).In certain embodiments, when two polypeptides are brought together (ortethered) by a bifunctional stapled peptide of the invention, they mightbe coming into such close molecular contact that one polypeptide (an“effector” biomolecule) might alter or modify the other polypeptide (a“target” biomolecule).

Structure of an Inventive Bifunctional Peptide

In certain embodiments, stapled or stitched bifunctional peptidescomprise three building blocks: A-L-E, comprising a targeting domain(A), a linker (L), and an effector domain (E) that are generallyarranged as follows:

wherein A and/or E is a stapled or stitched peptide, and L is a linker;wherein A is a targeting domain and E is an effector domain. A and E aretargeting or effector domains, that are sequences of amino acids thatmay or may not be stapled that specifically associate or bind topolypeptides, such as a target biomolecule or an effector biomolecule.Any part of the peptide A may be linked to any part of the peptide Ethrough the linker L. In certain embodiments, the linkage is N-terminusto N-terminus. In certain embodiments, the linkage is C-terminus toN-terminus. In certain embodiments, the linkage is C-terminus toC-terminus. In still other embodiments, the linkage may be throughinterior amino acids of one or both peptides. As will be appreciated byone on skill in the art, the linkage is typically positioned in such away as to avoid interfering with the binding activity of the peptide.The linkage may also be positioned in such a way to avoid interferingwith the stapling of the peptide.

In certain embodiments, where A is the targeting domain and specificallyassociates or binds to a target, E is the effector domain andspecifically associates or binds an effector biomolecule capable ofmodifying the target bound or associated with the targeting domain A. Lis a chemical linker that covalently links A and E. The linker L may bealiphatic or heteroaliphatic. In certain embodiments, linker L is 1-50atoms, in length, and may be optionally substituted. In certainembodiments, linker L is 1-25 atoms, in length, and may be optionallysubstituted.

A and E can have any length, that is they may comprise any number ofamino acids. The number of amino acids can be four or more, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,100 or more, or any number of amino acids in between 4 and 100. A and Ecan comprise a number of amino acids that is the minimal number of aminoacids sufficient to specifically bind or associate with either thetarget or the effector biomolecule. The amino acid sequence of one orboth of the domains may be substantially similar to or homologous to aknown peptide.

In one aspect, the present invention provides a bifunctional stapledpeptide wherein one or both domains comprise the formula:

wherein

each instance of L₁ and L₂ is, independently, a bond; cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedalkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkynylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted heteroalkenylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkynylene; substituted or unsubstituted arylene; substituted orunsubstituted heteroarylene; or substituted or unsubstituted acylene;

each instance of R^(a) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; cyclic or acyclic, substituted orunsubstituted acyl; or a suitable amino protecting group;

each instance of R^(b) is, independently, a suitable amino acid sidechain; hydrogen; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic oracyclic, substituted or unsubstituted acyl; substituted or unsubstitutedhydroxyl; substituted or unsubstituted thiol; substituted orunsubstituted amino; cyano; isocyano; halo; or nitro;

each instance of R^(e) is, independently, a bond to the linker moiety,—R^(E), —OR^(E), —N(R^(E))₂, or —SR^(E), wherein each instance of R^(E)is, independently, hydrogen; cyclic or acyclic, branched or unbranched,substituted or unsubstituted aliphatic; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; a resin; a suitable hydroxyl, amino, or thiolprotecting group; or two R^(E) groups of —N(R^(E))₂ together form asubstituted or unsubstituted 5- to 6-membered heterocyclic orheteroaromatic ring;

each instance of R^(f) is, independently, a bond to the linker moiety;hydrogen; cyclic or acyclic, branched or unbranched, substituted orunsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; a resin; a suitable amino protecting group; alabel optionally joined by a tether, wherein the tether is selected fromcyclic or acyclic, branched or unbranched, substituted or unsubstitutedalkylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkenylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkynylene; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroalkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkenylene; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted heteroalkynylene; substituted or unsubstituted arylene;substituted or unsubstituted heteroarylene; or substituted orunsubstituted acylene; or R^(f) and R^(a) of a terminal amino acidtogether form a substituted or unsubstituted 5- to 6-memberedheterocyclic or heteroaromatic ring;

each instance of R^(LL) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; substituted or unsubstituted acyl; substitutedor unsubstituted hydroxyl; substituted or unsubstituted thiol;substituted or unsubstituted amino; azido; cyano; isocyano; halo; nitro;

or two adjacent R^(LL) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring;

each instance of X_(AA) is, independently, a natural or unnatural aminoacid;

each instance of z is, independently, an integer between 2 to 6;

each instance of j is, independently, an integer between 1 to 10;

each instance of s and t is, independently, an integer between 0 and100;

each instance of q is, independently, an integer between 0 to 2; and

corresponds to a single or double bond.

In another aspect, the present invention provides a bifunctionalstitched peptide wherein one or both domains comprise the formula (i.e.;a peptide with multiple staples):

wherein

each instance of K, L₁, L₂, and M, is, independently, a bond; cyclic oracyclic, branched or unbranched, substituted or unsubstituted alkylene;cyclic or acyclic, branched or unbranched, substituted or unsubstitutedalkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkynylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted heteroalkenylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkynylene; substituted or unsubstituted arylene; substituted orunsubstituted heteroarylene; or substituted or unsubstituted acylene;

each instance of R^(a) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; cyclic or acyclic, substituted orunsubstituted acyl; or a suitable amino protecting group;

each instance of R^(b) is, independently, a suitable amino acid sidechain; hydrogen; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic oracyclic, substituted or unsubstituted acyl; substituted or unsubstitutedhydroxyl; substituted or unsubstituted thiol; substituted orunsubstituted amino; cyano; isocyano; halo; or nitro;

each instance of R^(e) is, independently, a bond to the linker moiety,—R^(E), —OR^(E), —N(R^(E))₂, or —SR^(E), wherein each instance of R^(E)is, independently, hydrogen; cyclic or acyclic, branched or unbranched,substituted or unsubstituted aliphatic; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; a resin; a suitable hydroxyl, amino, or thiolprotecting group; or two R^(E) groups of —N(R^(E))₂ together form asubstituted or unsubstituted 5- to 6-membered heterocyclic orheteroaromatic ring;

each instance of R^(f) is, independently, a bond to the linker moiety;hydrogen; cyclic or acyclic, branched or unbranched, substituted orunsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; a resin; a suitable amino protecting group; alabel optionally joined by a tether, wherein the tether is selected fromcyclic or acyclic, branched or unbranched, substituted or unsubstitutedalkylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkenylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkynylene; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroalkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkenylene; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted heteroalkynylene; substituted or unsubstituted arylene;substituted or unsubstituted heteroarylene; or substituted orunsubstituted acylene; or R^(f) and R^(a) together form a substituted orunsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring;

each instance of R^(KL), R^(LL), and R^(LM), is, independently,hydrogen; cyclic or acyclic, branched or unbranched, substituted orunsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; substituted or unsubstituted hydroxyl;substituted or unsubstituted thiol; substituted or unsubstituted amino;azido; cyano; isocyano; halo; nitro;

or two adjacent R^(KL) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring; two adjacent R^(KL) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring; or two adjacent R^(LM) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring;

each instance of X_(AA) is, independently, a natural or unnatural aminoacid;

each instance of y and z is, independently, an integer between 2 to 6;

each instance of j is, independently, an integer between 1 to 10;

each instance of p is, independently, an integer between 0 to 10;

each instance of s and t is, independently, an integer between 0 and100;

each instance of u, v, and q, is, independently, an integer between 0 to2; and

corresponds to a single or double bond.

In certain embodiments, one or both of peptides A and E is analpha-helical polypeptide. In certain embodiments, peptide A issubstantially alpha-helical. In certain embodiments, peptide E issubstantially alpha-helical. As used herein, the phrase “substantiallyalpha-helical” refers to a polypeptide adopting, on average, backbone(φ, ψ) dihedral angles in a range from about (−90°, −15°) to about(−35°, −70°). Alternatively, the phrase “substantially alpha-helical”refers to a polypeptide adopting dihedral angles such that the ψdihedral angle of one residue and the φ dihedral angle of the nextresidue sums, on average, about −80° to about −125°. In certainembodiments, the polypeptide adopts dihedral angles such that the ψdihedral angle of one residue and the φ dihedral angle of the nextresidue sums, on average, about −100° to about −110°. In certainembodiments, the polypeptide adopts dihedral angles such that the ψdihedral angle of one residue and the φ dihedral angle of the nextresidue sums, on average, about −105°. Furthermore, the phrase“substantially alpha-helical” may also refer to a polypeptide having atleast 50%, 60%, 70%, 80%, 90%, or 95% of the amino acids provided in thepolypeptide chain in an alpha-helical conformation, or with dihedralangles as specified herein. Confirmation of a polypeptide'salpha-helical secondary structure may be ascertained by known analyticaltechniques, such as x-ray crystallography, electron crystallography,fiber diffraction, fluorescence anisotropy, circular dichroism (CD), andnuclear magnetic resonance (NMR) spectroscopy.

The linker associating polypeptide A with polypeptide E may be anychemical moiety capable of associating the two polypeptides underconditions in which the bifunctional polypeptide will be used. Thelinker may be as simple as a covalent bond, or it may be a polymericlinker many atoms in length. In certain embodiments, the linker is apolypeptide or based on amino acids. In other embodiments, the linker isnot peptide like. In certain embodiments, the linker is a covalent bond(e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond,etc.). In certain embodiments, the linker is a carbon-nitrogen bond ofan amide linkage. In certain embodiments, the linker is a cyclic oracyclic, substituted or unsubstituted, branched or unbranched aliphaticor heteroaliphatic linker. In certain embodiments, the linker ispolymeric (e.g., polyethylene, polyethylene glycol, polyamide,polyester, etc.). In certain embodiments, the linker comprises amonomer, dimer, or polymer of aminoalkanoic acid. In certainembodiments, the linker comprises an aminoalkanoic acid (e.g., glycine,ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid,4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments,the linker comprises a monomer, dimer, or polymer of aminohexanoic acid(Ahx). In certain embodiments, the linker is based on a carbocyclicmoiety (e.g., cyclopentane, cyclohexane). In other embodiments, thelinker comprises a polyethylene glycol moiety (PEG). In otherembodiments, the linker comprises amino acids. In certain embodiments,the linker comprises a peptide. In certain embodiments, the linkercomprises an aryl or heteroaryl moiety. In certain embodiments, thelinker is based on a phenyl ring. In certain embodiments, the linkercomprises a triazole moiety (i.e., the product of a Huisgencycloaddition reaction). The linker may include functionalized moietiesto facilitate attachment of a nucleophile (e.g., thiol, amino) from thepeptide to the linker. Any electrophile may be used as part of thelinker. Exemplary electrophiles include, but are not limited to,activated esters, activated amides, Michael acceptors, alkyl halides,aryl halides, acyl halides, and isothiocyanates. In certain embodiments,the linker includes a maleimide group. In certain embodiments, thelinker includes a NHS ester. In certain embodiments, the linker includesboth a NHS ester and a maleimide group. For example, a cyclohexane ringmay be substituted with an NHS ester and a maleimide group. Examples ofcovalent conjugation strategies and suitable chemical conditions using avariety of linkers and/or functional groups to associate polypeptide Awith polypeptide E are set forth in FIGS. 27 to 32. In certainembodiments, thiol-maleimide conjugates are generated. In otherembodiments, 1,4- or 1,5-triazole conjugates are generated.

Uses of the Bifunctional Peptide

Bifunctional peptides of the invention may be used to tether twocellular entities together. In certain embodiments, by tethering twocellular entities, it is desired that one entity brings about a changein the other entity. One entity that brings about the change in theother entity is an effector biomolecule that modifies the other entity,which is the target. The modification of the target biomolecule somecharacteristic (e.g., biological activity) of the target. In someembodiments, by tethering two cellular entities, it is desired that thetwo entity are essentially irreversibly tethered together. For example,certain effector biomolecules may associate with a target or dissociatefrom a target naturally upon certain stimuli or molecular signals.Bifunctional peptides of the invention may be used to tether twocellular entities together irreversibly so that they do not dissociateupon such stimuli or other signals and remain associated. The effectorbiomolecule, for example, can be a cellular inhibitor of the target, ora particular molecular complex, that associates with the target to keepit in a certain intracellular localization, e.g. cytosolic or nuclear.In other embodiments bifunctional peptides can be used to tetherbiomolecules together that would only associate naturally upon certainstimuli or molecular signals, in the absence of such stimuli. In otherembodiments, biomolecules can be tethered together that do not naturallyassociate with each other. “Naturally” as used herein means in acellular context under physiological conditions including diseasedconditions.

In certain embodiments, bifunctional stapled peptides can be used toalter one or more characteristics of the target. In certain embodiments,the characteristics of the target are altered in such a way that thisalteration affects cell fate and/or cell behavior. In certainembodiments, changes in cell fate or cell behavior as a result ofchanges in one or more characteristics of the target affect the diseasestate of a subject, such as a mammal, for example, a human. In certainembodiments, bifunctional stapled peptides can be used to treat disease.In certain embodiments bifunctional stapled peptides can be used toprobe or elucidate biological pathways in research. The probing of abiological pathway can be performed both in vitro such as in cell ortissue culture, or in vivo, such as in an animal, e.g., humans, mice,rats, hamsters, fish, or primates.

In some embodiments, the two cellular entities are polypeptides, such asproteins and associated protein complexes. In certain embodiments,alterations or modifications of one entity (the target biomolecule) canbe the result of an enzymatic activity of the other entity (the effectormolecule). For proteins, for example, such alterations or modificationsmay comprise any of the posttranslational modifications known in theart.

Posttranslational modifications include, but are not limited to,ubiquitination, phosphorylation, acetylation, glycosylation,methylation, sumoylation, urmylation, neddylation, proteolysis,lipidation, acylation, famesylation, geranylgeranylation and/orligation. It will be appreciated by one of ordinary skill in the artthat posttranslational modifications can include the addition of achemical moiety as well as the removal of such a chemical moiety, asused herein, are any chemical groups (such as proteins, sugars, orinorganic molecules, for example phosphate) that can be added or removedto and from a polypeptide entity. Examples of such chemical moieties areproteins, such as ubiquitin (Ub), and ubiquitin-like proteins (Ubl)SUMO, Urml, and Nedd8, carbohydrates such as glycans, and small organicor inorganic groups such as phosphate, acetyl-, acyl-, or methyl-groups,or lipids. Chemical moieties may also include nucleic acids. Chemicalmoieties may be attached or removed from a target biomolecule by avariety of enzymatically active effector biomolecules.

In certain embodiments, the effector domain of the bifunctional peptiderecruits a ubiquitinating enzyme or ubiquitination machinery to a targetprotein. Ubiquitination of a target protein typically results indegradation of the ubiquitinated protein by the proteasome. Ubiquitin isattached to a protein, by ubiquitin ligases, such as E3 ligase. E3ligases can be single polypeptide chain enzymes, such as MDM2 or E6AP,or protein complexes, such as Skp1-Cullin-F-box (Skp1, Cul1/Cdc53,Roc1/Hrt/Rbx1, SCF), anaphase-promoting complex (APC), or BRCA1-Bard1complex. These complexes may associate with adaptor proteins, such ascdh1 and cdc20 for APC, and various F-box proteins for SCF, that providenatural substrate specificity, and further associate with E2 ubiquitinconjugating enzymes (UBCs).

Typically, ubiquitin is attached to a target protein through a series ofcatalytic steps. Ubiquitin is first activated by a ubiquitin activatingenzyme E1. The E1 enzyme catalyzes two reactions—ATP-dependentadenylation of the carboxylate followed by thiocarboxylate formationwith an internal cysteine of E1. This is followed by a trans-thiolationreaction that transfers Ub/Ubl to the active cysteine of the E2 enzyme.E2s then directly transfer the Ub/Ubl to the target lysine of the targetprotein, often aided by E3 ligase. Ub/Ubls can be transferred by afurther trans-thiolation reaction to HECT E3 ligases, which thentransfer the Ub/Ubl to substrates. In many cases multiple rounds ofubiquitination are catalyzed by a specialized E3 ligase resulting inpoly-Ub adducts.

Ubiquitins can be removed by de-ubiquitinating enzymes (DUBs), which areproteases. Examples of cysteine protease DUBs are: theubiquitin-specific processing protease (USP/UBP) superfamily; theubiquitin C-terminal hydrolyase (UCH) superfamily; the ovarian tumor(OTU) superfamily; and the Machado-Josephin domain (MJD) superfamily.

SUMO (small ubiquitin-related modifier) can be attached bysumoylation-dependent ubiquitin ligases, such as thesumoylation-dependent E3 ligases RanBP2, Pc2 and members of the PIASfamily. In humans and mice, the SUMO family consists of three members,SUMO-1, SUMO-2, and SUMO-3, which are encoded by separate genes. SUMOconjugation requires sequential E1-dependent activation, E2-dependentconjugation, and E3-dependent ligation steps. The human SUMO E1 enzymecomprises a heterodimer of the SAE1 and SAE2 proteins and forms athioester bond with glycine 97 of SUMO-1. Subsequently, SUMO-1 istransferred by transesterification to the SUMO-specific E2-conjugatingenzyme, Ubc9. Ubc9 can directly conjugate the carboxy terminal glycineof SUMO to lysines in target proteins that are situated in the consensusmotif yKxE/D, where y stands for valine, leucine, isoleucine,methionine, or phenylalanine; and x stands for any amino acid.

SUMO can be removed by desumoylation proteins or SUMO proteases, such asSuPrl, SENP1 (sentrin/SUMO-specific proteases), or ULPs (ubiquitin-likeprotein specific proteases).

Nedd8, a ubiquitin-like small protein modifier can be attached by aprocess called neddylation, which is similar to ubiquitination.Neddylation utilizes the E1 activating-enzyme complex composed of twosubunits, APP-BP1 and UBA3, and the E2 conjugating-enzyme, UBC12. Knownsubstrates of neddylation are Cullin family proteins: Cul1, Cul2, Cul3,Cul4A, Cul4B, and Cul5. Neddylation of certain cullins (e.g., Cullin-1),which are part of the SCF complexes might enhance E2-ubiquitinrecruitment to SCF and might be required for ubiquitination by SCF ofcertain E3 ligase substrates.

Deneddylation, which removes the Nedd8 moiety, might be accomplished byisopeptidase activity such as that of the COP9/signalosome (CSN) andCAND1.

In certain embodiments, the effector domain recruits an enzyme thatcatalyzes the acetylation of the of the target protein. Acetyl groupscan be attached by acetylases, such as, for example, PCAF/GCN5,p300/CBP, TAF250, SRC1 and MOZ, TIP60 or BRCA2, which may modify histoneand/or non-histone proteins. Acetylases may be part of large molecularweight complexes, such as TIP60, STAGA (SPT3-TAF9-GCN5/PCAF), ATAC (AdaTwo-A containing), or NuA4 histone acetylase complex or may associatewith transcriptions factors, such as, for example, E2Fs, TAFs, p53, andMyoD.

Acetyl groups can be removed by deacetylases, such as, for example,HDAC1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, that may modify histoneand/or non-histone proteins. HDACs may be part of large molecular weightcomplexes, such as NuRD, Sin3, SMRT, and N-CoR complex or may associatewith transcriptions factors, such as, for example, Rb, YY1, Sp1, MEF2,BRCA1, p53, c-Ski, and Ikaros.

In other embodiments, phosphorylation is typically accomplished bykinases. Kinases transfer a phosphate group from ATP or othernucleotides to the substrate via the side chain of an amino acid. Mostkinases transfer the phosphate group to serine or threonine (such as MAPkinases, ERK, JNK, and p38), others to tyrosine, and a number(dual-specificity kinases) to all three amino acids. There are alsoprotein kinases that phosphorylate other amino acids, includinghistidine kinases that phosphorylate histidine residues.

Dephosphorylation might be accomplished by phosphatases that remove aphosphate group from its substrate by hydrolyzing phosphoric acidmonoesters into a phosphate ion and a molecule with a free hydroxyl oramino group. Phosphatases include Cysteine-Dependent Phosphatases (CDPs)and metallo-phosphatases. Serine/threonine-specific protein phosphatasesinclude, for example, PP1 (α, β, γ1, γ2), PP2 (formerly 2A), PP3(formerly 2b, also known as calcineurin), PP2C, PP4, PP5, and PP6.Tyrosine-specific phosphatase include, for example, PTP1B. Dualspecificity phosphatases include, for example, VHR, DUSP1-DUSP28.Histidine Phosphatases include, for example, PHP. Lipid Phosphatasesinclude, for example, PTEN.

Methyl groups may be added to arginines of substrates by proteinmethylases, such as protein methylase I, II, or III; and PRMT1 and PRMT5(protein arginine methyltransferases 1 and 5), yielding for example,monomethyl or dimethyl arginines. Methylases can transfer methyl groupsusing, for example, S-adenosyl-L-methionine as a donor. Known proteinmethylation substrates include myelin basic protein (MBP), andheterogeneous ribonucleoprotein A1 (hnRNP A1). Methylases, known as HMT(histone methyl transferases), such as, for example, SUV39H1, G9a,EHMT1, Trithorax, Ash1 and Dot1, or other enzymes, modify primarilyhistone proteins, particularly on lysine and arginine residues resultingin mono-, di-, or tri-methylated substrates.

Demethylation might be accomplished by demethylases, such as LSD1, JMJD,or JHDM. Known targets of demethylases are for example histone proteins.

Carbohydrate moieties can be added to proteins or lipids byglycosyltransferases, such as GlcNAc-transferase (GnTI, II, II, IV, V),galactosyltransferase, glucuronyltransferase, sialyltransferase,xylosyltransferase, fucosyltransferase, and mannosyltransferase.Glycosyltransferases transfer a monosaccharide unit from an activatedsugar phosphate to an acceptor molecule, for example, tyrosine, serine,or threonine to give O-linked glycoproteins, or to asparagine to giveN-linked glycoproteins. Mannosyl groups may be transferred to tryptophanto generate C-mannosyl tryptophan. The result of glycosyl transfer canbe a monosaccharide glycoside, an oligosaccharide, or a polysaccharide.Common donors for glycosyltransferases are, for example, UDP-glucose,UDP-galactose, UDP-GlcNAc, UDP-GalNAc, UDP-xylose, UDP-glucuronic acid,GDP-mannose, GDP-fucose, and CMP-sialic acid. Lipid linked glycosyldonors can also be used, where the lipid is frequently a terpenoid suchas dolichol or polyprenol.

Carbohydrates can be removed from proteins or lipids by glycosidases(glycoside hydrolases) that catalyze the hydrolysis of the glycosidiclinkage of sugars.

Prenylation is a lipid posttranslational modification of proteins whichtypically occurs at a cysteine residue. The specific sequence recognizedby prenyltransferases consists either of the CaaX box forfamesyltransferase (FTase) and geranylgeranyltransferase 1 (GGTase1) orC-terminal cysteines of Rab GTPases in the case ofgeranylgeranyltransferase 2 (GGTase2). The anchor can be of farnesyl (3isoprenyl units) or of geranylgeranyl (4 isoprenyl units) type. Thismodification allows membrane attachment or association of thepyrenylated protein. Farnesylation involves the enzymefarnesyltransferase (FTase) transferring a farnesyl group from farnesylpyrophosphate (FPP) to a substrate, e.g., Ras protein. A related enzyme,geranylgeranyltransferase I (GGTase I), transfers a geranylgeranyl groupto the substrate, e.g., K and N-Ras.

It will be appreciated by one of ordinary skill that any of themodification described herein may occur at one or more sites (or targetamino acids) on the target biomolecule. For example, a target entity canbe phosphorylated at multiple sites, that is one, two, three, four ormore sites. Chemical moieties can be attached as monomers or multimersto the same site, for ubiquitin, and the same amino acid target site canbe modified one or more times with the same chemical moiety, such as,for example, mono-, di-, or tri-methylation.

It should further be appreciated that addition, removal, or replacementof chemical moieties may have different effects on the targetbiomolecule. Many of these effects are well known in the art. Some ofthe modifications can be additive, for example, in activating a targetbiomolecule to carry out a certain function, some modifications may haveopposing effects, for example protecting a substrate from or marking itfor degradation.

For example, SUMO-1 and ubiquitin can compete for the same sites on atarget biomolecule and can have opposing effects. Poly-ubiquitination ofthe target biomolecule can lead to degradation by the 26S proteasome,while SUMOylation can protect the target biomolecule from degradation(this is known, for example for IkappaB). DNA damage-induced acetylationon specific sites of the tumor suppressor protein Rb can preventphosphorylation on these sites (e.g., by CDKs) and keeping Rb in anactive conformation. Phosphorylation of specific sites of the E2F1transcription factor (e.g., by ATM/ATR) promotes E2F1 acetylation (e.g.,by CBP/p/CAF).

High expression of some glycosyl epitopes promotes invasion andmetastasis, such as, for example, β6GlcNAc branching in N-linkedstructure; sialyl-Tn in O-linked structure; sialyl-Lex, sialyl-Lea, andLey in either N-linked, O-linked, or lipid-linked structure; GM2, GD3,and sialyl-Gb5 in lipid-linked structure. High expression of otherglycosyl epitopes suppresses tumor progression, such as, for example,β4GlcNAc competitive with β6GlcNAc; histo-blood group A and Bcompetitive with sialylated structures including sialyl-Lex andsialyl-Lea; Gb5 competitive with sialyl-Gb5.

For example, one common glycosylation change associated with malignancyis enhanced β6GlcNAc side chain branching of N-linked structure, causedby enhanced activity of GnT-V, and counteracting β4GlcNAc (bisectingGlcNAc) synthesized by GnT-III. The level of both glycosyl epitopes isdetermined by the balance between GnT-V and GnT-III. Enhanced GnT-IIIgene can inhibit β6GlcNAc branching, which can lead to suppression ofmetastasis. In metastasis, one of the targets appears to be E-cadherin,in which enhanced β4GlcNAc reduces β6GlcNAc branching, leading toenhanced cadherin-dependent cell-to-cell adhesion and consequentsuppression of metastasis. A bifunctional peptide of the inventioncomprising an effector domain that specifically binds to GnT-III and atargeting domain that specifically binds to E-cadherin is used to tetherGnTIII to E-cadherin in cancer cells, leading to increased β4GlcNAcmodification on E-cadherin and suppression of metastasis.

Further, it is well known in the art that certain modifications resultin the subsequent recruitment of additional enzymes to the modifiedtarget biomolecule that add or remove other modifications. For example,p53 accumulation and activation are regulated through posttranslationalmodifications such as phosphorylation, acetylation and ubiquitination.Phosphorylation of Ser15, Thr18, Ser20 and Ser37 stabilizes p53 bydisrupting interaction between p53 and MDM2, whereas phosphorylation atthe p53 C-terminus such as at Ser315 and Ser392 are reported to regulatethe oligomerization state and sequence-specific DNA binding ability ofp53.

The PI3K family including ATM (ataxia telangiectasia mutated) and ATR(ATM- and Rad3-related) are mainly responsible for p53 phosphorylation.ATM mainly phosphorylates the Ser15 residue in response to irradiationand chemotherapeutic drugs, while ATR especially phosphorylates bothSer15 and Ser37 residues in response to UV and inhibitors ofreplication.

Phosphorylation of p53 leads to subsequent acetylation. For example,phosphorylation at N-terminal serines, such as Ser15, Ser33 and Ser37recruits p300/CBP and PCAF to induce p53 acetylation in response to DNAdamage. Phosphorylation of p53 at Ser20 or Thr18 can stabilize thep300-p53 complex and thus induce p53 acetylation. p300/CBP acetylatesp53 at K305, K372, K373, K381 and K382, whereas PCAF acetylates p53 atK320. Tip60 specifically acetylates p53 at K120 in response to DNAdamage. p53 acetylation can increase p53 sequence-specific DNA-bindingcapacity or enhance its stabilization by inhibiting ubiquitination ofp53 mediated by MDM2.

A bifunctional peptide of the invention comprising an effector domainthat specifically binds to ATM or ATR and a targeting domain thatspecifically binds to p53 is used to tether the PI3 kinase to p53 incancer cells, in which p53 is not fully inactivated as a result ofmutations, to promote phosphorylation and subsequent p53 acetylation,leading to stabilization and activation of p53.

A bifunctional peptide of the invention comprising an effector domainthat specifically binds to p300 or PCAF and a targeting domain thatspecifically binds to p53 is used to tether the acetylase to p53 incancer cells, in which p53 is not fully inactivated as a result ofmutations, to promote p53 acetylation, leading to stabilization andactivation of p53.

In some embodiments, the effector biomolecule adds, removes or replacesone or more chemical moieties on one or more amino acid sites of thetarget biomolecule. In certain embodiments, the modification leads to adesired change in fate of the target biomolecule, such as activation,de-activation, changes in intracellular localization, stabilization,de-stabilization, changes in substrate specificity or enzyme fidelity,or changes in protein folding of the target biomolecule.

It should therefore be appreciated that by controlling one or morespecific posttranslational modifications one can control manycharacteristics of a target biomolecule, such as, for example, theenzymatic activity, substrate specificity, intra-cellular localization,degradation, half-life, localization, protein-protein interaction,protein-nucleic acid interaction, and stability of the targetbiomolecule. In certain embodiments, these changes in fate of the targetbiomolecule lead to a change in the fate of the cell harboring thetarget biomolecule.

In certain embodiments, bifunctional stapled peptides of the inventioncan be used to tether an effector biomolecule with a target biomoleculeto modify the folding state of the target biomolecule. For example,certain nascent polypeptide chains can encounter problems during theprotein folding process. Improperly folded or misfolded proteins mightlose some or all of their activity, might gain ectopic activities, mightbe mislocalized within the cell or might form disruptive proteinaggregates. Misfolded proteins are known to be involved in thedevelopment of many diseases, particularly neuronal or brain diseases.For example, misfolded alpha-synuclein is associated with Parkinson'sDisease.

In certain embodiments, bifunctional stapled peptides of the inventioncan be used to tether chaperones to misfolded proteins or proteins atrisk of misfolding. Chaperones can also be tethered to protein complexesto aid complex formation. There are several families of chaperones, suchas, for example, 40-kDa heat shock protein (HSP40; DnaJ), 60-kDa heatshock protein (HSP60; GroEL), 70-kDa heat shock protein (HSP70; DnaK),90-kDa heat shock protein (HSP90; HtpG), and 100-kDa heat shock protein(HSP100; Clp). Other chaperones include BiP, GRP94, GRP170, calnexin,calreticulin, HSP47, ERp29, protein disulfide isomerase, peptidyl prolylcis-trans-isomerase, and ERp57.

The present invention provides a method of treating a disease, disorder,or condition comprising administering to a subject diagnosed with orhaving susceptibility to the disease, disorder, or condition, atherapeutically effective amount of an inventive bifunctionalpolypeptide, or pharmaceutically acceptable form thereof. Exemplarydiseases, disorders, or conditions which may be treated byadministration of an inventive bifunctional polypeptide compriseproliferative, neurological, immunological, endocrinologic,cardiovascular, hematologic, and inflammatory diseases, disorders, orconditions, and conditions characterized by premature or unwanted celldeath.

As used herein a proliferative disease, condition, or disorder includes,but is not limited to, cancer, hematopoietic neoplastic disorders,proliferative breast disease, proliferative disorders of the lung,proliferative disorders of the colon, proliferative disorders of theliver, and proliferative disorders of the ovary.

Examples of cancers treatable by the above method include carcinoma,sarcoma, or metastatic disorders, breast cancer, ovarian cancer, coloncancer, lung cancer, fibrosarcoma, myosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer,pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer,cancer of the head and neck, skin cancer, brain cancer, squamous cellcarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, testicular cancer, small cell lung carcinoma, non-small celllung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi'ssarcoma.

Examples of hematopoietic neoplastic disorders treatable by the abovemethod includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. In certain embodiments, thediseases arise from poorly differentiated acute leukemias, e.g.,erythroblastic leukemia and acute megakaryoblastic leukemia. Additionalexemplary myeloid disorders include, but are not limited to, acutepromyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stembergdisease.

Examples of proliferative breast disease treatable by the above methodincludes epithelial hyperplasia, sclerosing adenosis, and small ductpapillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodestumor, and sarcomas, and epithelial tumors such as large duct papilloma;carcinoma of the breast including in situ (noninvasive) carcinoma thatincludes ductal carcinoma in situ (including Paget's disease) andlobular carcinoma in situ, and invasive (infiltrating) carcinomaincluding, but not limited to, invasive ductal carcinoma, invasivelobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma,tubular carcinoma, and invasive papillary carcinoma, and miscellaneousmalignant neoplasms. Disorders in the male breast include, but are notlimited to, gynecomastia and carcinoma.

Examples of proliferative disorders of the lung treatable by the abovemethod include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, non-inflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of proliferative disorders of the colon treatable by the abovemethod include, but are not limited to, non-neoplastic polyps, adenomas,familial syndromes, colorectal carcinogenesis, colorectal carcinoma, andcarcinoid tumors.

Examples of proliferative disorders of the liver treatable by the abovemethod include, but are not limited to, nodular hyperplasias, adenomas,and malignant tumors, including primary carcinoma of the liver andmetastatic tumors.

Examples of proliferative disorders of the ovary treatable by the abovemethod include, but are not limited to, ovarian tumors such as, tumorsof coelomic epithelium, serous tumors, mucinous tumors, endometeriodtumors, clear cell adenocarcinoma, cystadenofibroma, brenner tumor,surface epithelial tumors; germ cell tumors such as mature (benign)teratomas, monodermal teratomas, immature malignant teratomas,dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomaltumors such as, granulosa-theca cell tumors, thecomafibromas,androblastomas, hill cell tumors, and gonadoblastoma; and metastatictumors such as Krukenberg tumors.

The bifunctional polypeptides described herein can also be used totreat, prevent or diagnose conditions characterized by overactive celldeath or cellular death due to physiologic insult, etc. Some examples ofconditions characterized by premature or unwanted cell death are oralternatively unwanted or excessive cellular proliferation include, butare not limited to hypocellular/hypoplastic, acellular/aplastic, orhypercellular/hyperplastic conditions. Some examples include hematologicdisorders including but not limited to fanconi anemia, aplastic anemia,thalaessemia, congenital neutropenia, myelodysplasia. The polypeptidesof the invention that act to decrease apoptosis can be used to treatdisorders associated with an undesirable level of cell death. Thus, theanti-apoptotic peptides of the invention can be used to treat disorderssuch as those that lead to cell death associated with viral infection,e.g., infection associated with infection with human immunodeficiencyvirus (HIV).

A wide variety of neurological diseases are characterized by the gradualloss of specific sets of neurons, and the anti-apoptotic peptides can beused in the treatment of these disorders. Such disorders includeAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis(ALS) retinitis pigmentosa, spinal muscular atrophy, and various formsof cerebellar degeneration. The cell loss in these diseases does notinduce an inflammatory response, and apoptosis appears to be themechanism of cell death. In addition, a number of hematologic diseasesare associated with a decreased production of blood cells. Thesedisorders include anemia associated with chronic disease, aplasticanemia, chronic neutropenia, and the myelodysplastic syndromes.Disorders of blood cell production, such as myelodysplastic syndrome andsome forms of aplastic anemia, are associated with increased apoptoticcell death within the bone marrow. These disorders could result from theactivation of genes that promote apoptosis, acquired deficiencies instromal cells or hematopoietic survival factors, or the direct effectsof toxins and mediators of immune responses. Two common disordersassociated with cell death are myocardial infarctions and stroke. Inboth disorders, cells within the central area of ischemia, which isproduced in the event of acute loss of blood flow, appear to die rapidlyas a result of necrosis. However, outside the central ischemic zone,cells die over a more protracted time period and morphologically appearto die by apoptosis. The anti-apoptotic peptides of the invention can beused to treat all such disorders associated with undesirable cell death.

Some examples of neurologic disorders that can be treated with thebifunctional polypeptides described herein include but are not limitedto Alzheimer's Disease, Down's Syndrome, Dutch Type Hereditary CerebralHemorrhage Amyloidosis, Reactive Amyloidosis, Familial AmyloidNephropathy with Urticaria and Deafness, Muckle-Wells Syndrome,Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, FamilialAmyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, IsolatedCardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes,Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid,Familial Amyloidosis, Hereditary Cerebral Hemorrhage With Amyloidosis,Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob disease,Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis,a Prion-mediated disease, Huntington's disease, Pick's disease,Amyotrophic Lateral Schlerosis (ALS), Parkinson's disease, and Lewy BodyDisease.

Some examples of endocrinologic disorders that can be treated with thebifunctional polypeptides described herein include but are not limitedto diabetes, hypothyroidism, hypopituitarism, hypoparathyroidism,hypogonadism, fertility disorders, etc.

Some examples of immunologic disorders that can be treated with thepolypeptides described herein include but are not limited to organtransplant rejection, arthritis, lupus, IBD, Crohn's disease, asthma,multiple sclerosis, diabetes, Graft versus host diseases, autoimmunediseases, psoriasis, rheumatoid arthritis, etc.

Examples of cardiovascular disorders that can be treated or preventedwith the polypeptides of the invention include, but are not limited to,atherosclerosis, myocardial infarction, stroke, thrombosis, aneurism,heart failure, ischemic heart disease, angina pectoris, sudden cardiacdeath, hypertensive heart disease; non-coronary vessel disease, such asarteriolosclerosis, small vessel disease, nephropathy,hypertriglyceridemia, hypercholesterolemia, hyperlipidemia,xanthomatosis, asthma, hypertension, emphysema and chronic pulmonarydisease; or a cardiovascular condition associated with interventionalprocedures (“procedural vascular trauma”), such as restenosis followingangioplasty, placement of a shunt, stent, synthetic or natural excisiongrafts, indwelling catheter, valve or other implantable devices.

The inventive bifunctional polypeptides may serve to treat theabove-described diseases, disorders, or conditions, by tetheringcellular entities, such as proteins, together, as described herein.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprising aninventive bifunctional polypeptide, or a pharmaceutically acceptableform thereof, and a pharmaceutically acceptable excipient. Suchpharmaceutical compositions may optionally comprise one or moreadditional biologically active substances. In accordance with someembodiments, a method of administering a pharmaceutical compositioncomprising inventive compositions to a subject in need thereof isprovided. In some embodiments, inventive compositions are administeredto humans. For the purposes of the present invention, the phrase “activeingredient” generally refers to an inventive bifunctional polypeptide,as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and/or other primates; mammals, includingcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,and/or dogs; and/or birds, including commercially relevant birds, suchas chickens, ducks, geese, and/or turkeys.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in bulk, as a single unit dose, and/or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

Pharmaceutical formulations of the present invention may additionallycomprise a pharmaceutically acceptable excipient, which, as used herein,includes any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md.,2006) discloses various excipients used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional excipient medium is incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention.

In some embodiments, the pharmaceutically acceptable excipient is atleast 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, theexcipient is approved for use in humans and for veterinary use. In someembodiments, the excipient is approved by United States Food and DrugAdministration. In some embodiments, the excipient is pharmaceuticalgrade. In some embodiments, the excipient meets the standards of theUnited States Pharmacopoeia (USP), the European Pharmacopoeia (EP), theBritish Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in the inventive formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpolyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers {e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays {e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminumsilicate]), long chain amino acid derivatives, high molecular weightalcohols {e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers {e.g.,carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives {e.g.,carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters {e.g., polyoxyethylenesorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60],polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate[Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span65], glyceryl monooleate, sorbitan monooleate [Span 80]),polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., Cremophor), polyoxyethyleneethers, (e.g., polyoxyethylene lauryl ether [Brij 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.,cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g., acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,polyvinylpyrrolidone), magnesium aluminum silicate (Veegum), and larcharabogalactan); alginates; polyethylene oxide; polyethylene glycol;inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;alcohol; etc.; and combinations thereof.

Exemplary preservatives may include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives. Exemplaryantioxidants include, but are not limited to, alpha tocopherol, ascorbicacid, acorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, monothioglycerol, potassium metabisulfite, propionicacid, propyl gallate, sodium ascorbate, sodium bisulfite, sodiummetabisulfite, and sodium sulfite. Exemplary chelating agents includeethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malicacid, phosphoric acid, sodium edetate, tartaric acid, and trisodiumedetate. Exemplary antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservativesinclude, but are not limited to, vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid. Other preservatives include, but arenot limited to, tocopherol, tocopherol acetate, deteroxime mesylate,cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ethersulfate (SLES), sodium bisulfite, sodium metabisulfite, potassiumsulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben,Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certainembodiments, the preservative is an anti-oxidant. In other embodiments,the preservative is a chelating agent.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., andcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,Litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthom, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredients, the liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions caninclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents. In certainembodiments for parenteral administration, the conjugates of theinvention are mixed with solubilizing agents such as CREMOPHOR,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the conjugates of thisinvention with suitable non-irritating excipients, such as cocoa butter,polyethylene glycol or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active ingredients can be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient may be admixed with at least oneinert diluent such as sucrose, lactose or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of aconjugate of this invention may include ointments, pastes, creams,lotions, gels, powders, solutions, sprays, inhalants and/or patches.Generally, the active component is admixed under sterile conditions witha pharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present inventioncontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of an active ingredient tothe body. Such dosage forms may be prepared, for example, by dissolvingand/or dispensing the active ingredient in the proper medium.Alternatively or additionally, the rate may be controlled by eitherproviding a rate controlling membrane and/or by dispersing the activeingredient in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in a formulation suitable for pulmonary administration viathe buccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers or from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder and/or using a self propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may provide the active ingredient in the form of droplets of asolution and/or suspension. Such formulations may be prepared, packaged,and/or sold as aqueous and/or dilute alcoholic solutions and/orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization and/oratomization device. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, a flavoring agentsuch as saccharin sodium, a volatile oil, a buffering agent, a surfaceactive agent, and/or a preservative such as methylhydroxybenzoate. Thedroplets provided by this route of administration may have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare useful for intranasal delivery of a pharmaceutical composition ofthe invention. Another formulation suitable for intranasaladministration is a coarse powder comprising the active ingredient andhaving an average particle from about 0.2 to 500 micrometers. Such aformulation is administered in the manner in which snuff is taken, i.e.,by rapid inhalation through the nasal passage from a container of thepowder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition of theinvention may be prepared, packaged, and/or sold in a formulationsuitable for buccal administration. Such formulations may, for example,be in the form of tablets and/or lozenges made using conventionalmethods, and may, for example, 0.1 to 20% (w/w) active ingredient, thebalance comprising an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising the active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 to about 200nanometers, and may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in a formulation suitable for ophthalmic administration.Such formulations may, for example, be in the form of eye dropsincluding, for example, a 0.1/1.0% (w/w) solution and/or suspension ofthe active ingredient in an aqueous or oily liquid excipient. Such dropsmay further comprise buffering agents, salts, and/or one or more otherof the additional ingredients described herein. Otherophthalmically-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form and/or ina liposomal preparation. Ear drops and/or eye drops are contemplated asbeing within the scope of this invention.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005.

Administration

In some embodiments, a therapeutically effective amount of an inventivepharmaceutical composition is delivered to a patient and/or organismprior to, simultaneously with, and/or after diagnosis with a disease,disorder, and/or condition. In some embodiments, a therapeutic amount ofan inventive composition is delivered to a patient and/or organism priorto, simultaneously with, and/or after onset of symptoms of a disease,disorder, and/or condition. In some embodiments, the amount of inventiveconjugate is sufficient to treat, alleviate, ameliorate, relieve, delayonset of, inhibit progression of, reduce severity of, and/or reduceincidence of one or more symptoms or features of the disease, disorder,and/or condition.

The compositions, according to the method of the present invention, maybe administered using any amount and any route of administrationeffective for treatment. The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the infection, the particularcomposition, its mode of administration, its mode of activity, and thelike. The compositions of the invention are typically formulated indosage unit form for ease of administration and uniformity of dosage. Itwill be understood, however, that the total daily usage of thecompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular subject ororganism will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific active ingredient employed; the specific composition employed;the age, body weight, general health, sex and diet of the subject; thetime of administration, route of administration, and rate of excretionof the specific active ingredient employed; the duration of thetreatment; drugs used in combination or coincidental with the specificactive ingredient employed; and like factors well known in the medicalarts.

The pharmaceutical compositions of the present invention may beadministered by any route. In some embodiments, the pharmaceuticalcompositions of the present invention are administered variety ofroutes, including oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, subcutaneous, intraventricular,transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical(as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal,enteral, sublingual; by intratracheal instillation, bronchialinstillation, and/or inhalation; and/or as an oral spray, nasal spray,and/or aerosol. Specifically contemplated routes are systemicintravenous injection, regional administration via blood and/or lymphsupply, and/or direct administration to an affected site. In general themost appropriate route of administration will depend upon a variety offactors including the nature of the agent (e.g., its stability in theenvironment of the gastrointestinal tract), the condition of the subject(e.g., whether the subject is able to tolerate oral administration),etc. At present the oral and/or nasal spray and/or aerosol route is mostcommonly used to deliver therapeutic agents directly to the lungs and/orrespiratory system. However, the invention encompasses the delivery ofthe inventive pharmaceutical composition by any appropriate route takinginto consideration likely advances in the sciences of drug delivery.

In certain embodiments, the bifunctional polypeptides of the inventionmay be administered at dosage levels sufficient to deliver from about0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg,from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg toabout 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject bodyweight per day, one or more times a day, to obtain the desiredtherapeutic effect. The desired dosage may be delivered three times aday, two times a day, once a day, every other day, every third day,every week, every two weeks, every three weeks, or every four weeks. Incertain embodiments, the desired dosage may be delivered using multipleadministrations (e.g., two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, or more administrations).

It will be appreciated that inventive bifunctional polypeptides andpharmaceutical compositions of the present invention can be employed incombination therapies. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. It will beappreciated that the therapies employed may achieve a desired effect forthe same purpose (for example, an inventive conjugate useful fordetecting tumors may be administered concurrently with another agentuseful for detecting tumors), or they may achieve different effects(e.g., control of any adverse effects).

Pharmaceutical compositions of the present invention may be administeredeither alone or in combination with one or more other therapeuticagents. By “in combination with,” it is not intended to imply that theagents must be administered at the same time and/or formulated fordelivery together, although these methods of delivery are within thescope of the invention. The compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. In general, each agent will beadministered at a dose and/or on a time schedule determined for thatagent. Additionally, the invention encompasses the delivery of theinventive pharmaceutical compositions in combination with agents thatmay improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body.

The particular combination of therapies (therapeutics and/or procedures)to employ in a combination regimen will take into account compatibilityof the desired therapeutics and/or procedures and/or the desiredtherapeutic effect to be achieved. It will be appreciated that thetherapies employed may achieve a desired effect for the same disorder(for example, an inventive polypeptide may be administered concurrentlywith another biologically active agent used to treat the same disorder),and/or they may achieve different effects (e.g., control of any adverseeffects). In some embodiments, polypeptides of the invention areadministered with a second biologically active agent that is approved bythe U.S. Food and Drug Administration.

In will further be appreciated that biologically active agents utilizedin this combination may be administered together in a single compositionor administered separately in different compositions.

In general, it is expected that biologically active agents utilized incombination be utilized at levels that do not exceed the levels at whichthey are utilized individually. In some embodiments, the levels utilizedin combination will be lower than those utilized individually.

In some embodiments, inventive pharmaceutical compositions may beadministered in combination with any biologically active agent ortherapeutic regimen that is useful to treat, alleviate, ameliorate,relieve, delay onset of, inhibit progression of, reduce severity of,and/or reduce incidence of one or more symptoms or features of cancer.For example, inventive compositions may be administered in combinationwith traditional cancer therapies including, but not limited to,surgery, chemotherapy, radiation therapy, hormonal therapy,immunotherapy, complementary or alternative therapy, and any combinationof these therapies.

In some embodiments, inventive compositions are administered incombination with surgery to remove a tumor. Because complete removal ofa tumor with minimal or no damage to the rest of a patient's body istypically the goal of cancer treatment, surgery is often performed tophysically remove part or all of a tumor. If surgery is unable tocompletely remove a tumor, additional therapies (e.g., chemotherapy,radiation therapy, hormonal therapy, immunotherapy, complementary oralternative therapy) may be employed.

In some embodiments, inventive compositions are administered incombination with radiation therapy. Radiation therapy (also known asradiotherapy, X-ray therapy, or irradiation) is the use of ionizingradiation to kill cancer cells and shrink tumors. Radiation therapy maybe used to treat almost any type of solid tumor, including cancers ofthe brain, breast, cervix, larynx, lung, pancreas, prostate, skin,stomach, uterus, or soft tissue sarcomas. Radiation can be used to treatleukemia and lymphoma. Radiation therapy can be administered externallyvia external beam radiotherapy (EBRT) or internally via brachytherapy.Typically, the effects of radiation therapy are localized and confinedto the region being treated. Radiation therapy injures or destroys tumorcells in an area being treated (e.g., a target organ, tissue, and/orcell) by damaging their genetic material, preventing tumor cells fromgrowing and dividing. In general, radiation therapy attempts to damageas many tumor cells as possible while limiting harm to nearby healthytissue. Hence, it is often administered in multiple doses, allowinghealthy tissue to recover between fractions.

In some embodiments, inventive compositions are administered incombination with immunotherapy. Immunotherapy is the use of immunemechanisms against tumors which can be used in various forms of cancer,such as breast cancer (e.g., trastuzumab/Herceptin®), leukemia (e.g.,gemtuzumab ozogamicin/Mylotarg®), and non-Hodgkin's lymphoma (e.g.,rituximab/Rituxan®). In some embodiments, immunotherapy agents aremonoclonal antibodies directed against proteins that are characteristicto the cells of the cancer in question. In some embodiments,immunotherapy agents are cytokines that modulate the immune system'sresponse. In some embodiments, immunotherapy agents may be vaccines.

In some embodiments, vaccines can be administered to prevent and/ordelay the onset of cancer. In some embodiments, cancer vaccines preventand/or delay the onset of cancer by preventing infection by oncogenicinfectious agents. In some embodiments, cancer vaccines prevent and/ordelay the onset of cancer by mounting an immune response againstcancer-specific epitopes. To give but one example of a cancer vaccine,an experimental vaccine for HPV types 16 and 18 was shown to be 100%successful at preventing infection with these types of HPV and, thus,are able to prevent the majority of cervical cancer cases (Harper etal., 2004, Lancet, 364:1757).

In some embodiments, inventive compositions are administered incombination with complementary and alternative medicine treatments. Someexemplary complementary measures include, but are not limited to,botanical medicine (e.g., use of mistletoe extract combined withtraditional chemotherapy for the treatment of solid tumors); acupuncturefor managing chemotherapy-associated nausea and vomiting and incontrolling pain associated with surgery; prayer; psychologicalapproaches (e.g., “imaging” or meditation) to aid in pain relief orimprove mood. Some exemplary alternative measures include, but are notlimited to, diet and other lifestyle changes (e.g., plant-based diet,the grape diet, and the cabbage diet).

In some embodiments, inventive compositions are administered incombination with any of the traditional cancer treatments describedherein, which are often associated with unpleasant, uncomfortable,and/or dangerous side effects. For example, chronic pain often resultsfrom continued tissue damage due to the cancer itself or due to thetreatment (i.e., surgery, radiation, chemotherapy). Alternatively oradditionally, such therapies are often associated with hair loss,nausea, vomiting, diarrhea, constipation, anemia, malnutrition,depression of immune system, infection, sepsis, hemorrhage, secondaryneoplasms, cardiotoxicity, hepatotoxicity, nephrotoxicity, ototoxicity,etc. Thus, inventive compositions which are administered in combinationwith any of the traditional cancer treatments described herein may bealso be administered in combination with any therapeutic agent ortherapeutic regimen that is useful to treat, alleviate, ameliorate,relieve, delay onset of, inhibit progression of, reduce severity of,and/or reduce incidence of one or more side effects of cancer treatment.To give but a few examples, pain can be treated with opioids and/oranalgesics (e.g., morphine, oxycodone, antiemetics, etc.); nausea andvomiting can be treated with 5-HT₃ inhibitors (e.g.,dolasetron/Anzemet®, granisetron/Kytril®, ondansetron/Zofran®,palonsetron/Aloxi®) and/or substance P inhibitors (e.g.,aprepitant/Emend®); immunosuppression can be treated with a bloodtransfusion; infection and/or sepsis can be treated with antibiotics(e.g., penicillins, tetracyclines, cephalosporins, sulfonamides,aminoglycosides, etc.); and so forth.

In some embodiments, inventive compositions may be administered and/orinventive diagnostic methods may be performed in combination with anytherapeutic agent or therapeutic regimen that is useful to diagnose oneor more symptoms or features of cancer (e.g., detect the presence ofand/or locate a tumor). In some embodiments, inventive conjugates may beused in combination with one or more other diagnostic agents. To givebut one example, conjugates used to detect tumors may be administered incombination with other agents useful in the detection of tumors. Forexample, inventive conjugates may be administered in combination withtraditional tissue biopsy followed by immunohistochemical staining andserological tests (e.g., prostate serum antigen test). Alternatively oradditionally, inventive conjugates may be administered in combinationwith a contrasting agent for use in computed tomography (CT) scansand/or MRI.

Methods of Preparing and Synthesizing Bifunctional Stapled or StitchedPeptides

In certain embodiments, the targeting and effector domains A and E ofthe bifunctional stapled or stitched peptides of the invention aredesigned and synthesized de novo. In certain embodiments, the targetingand effector domains A and E comprise one or more non-natural aminoacids (Tables 1 and 2). In certain embodiments, the targeting andeffector domains A and E can be stapled or stitched, as describedherein, by crosslinking moieties to stabilize the secondary structure ofthe A and E domains.

In general, the synthesis of these stabilized secondary structuresinvolves (1) synthesizing a peptide from a selected number of natural ornon-natural amino acids, wherein said peptide comprises at least tworeactive moieties capable of undergoing a C—C bond forming reaction; and(2) contacting said peptide with a reagent to generate at least onecrosslinker and to effect stabilization of a specific secondarystructure motif (e.g., an α-helix).

As one of ordinary skill in the art will realize, the number,stereochemistry, and type of amino acid structures (natural ornon-natural) selected will depend upon the size and shape of thesecondary structure to be prepared (e.g., length of an α-helix), theability of the particular amino acids to generate a secondary structuralmotif that are desirable to mimic. The secondary structure to beprepared depends on the desired biological activity, that is the abilityto target an effector biomolecule or a target biomolecule with anaffinity sufficient to be specific and to follow the two biomoleculestogether.

It will be appreciated, that the number of crosslinking moieties is notlimited to one or two, rather the number of crosslinking moietiesutilized can be varied with the length of the targeting and/or effectordomain as desired, and as compatible with the desired structure andactivity to be generated.

The synthesis of an inventive bifunctional polypeptide first involvesthe selection of a desired sequence and number of amino acids and aminoacid analogues. As one of ordinary skill in the art will realize, thenumber, stereochemistry, and type of amino acid structures (natural ornon-natural) selected will depend upon the size of the polypeptide to beprepared, the ability of the particular amino acids to generate adesired structural motif (e.g., an alpha-helix), and any particularmotifs that are desirable to mimic to generate protein domains thateffectively bind to the target or effector biomolecule.

Once the amino acids are selected, synthesis of the inventivepolypeptide can be achieved using standard deprotection and couplingreactions. Formation of peptide bonds and polypeptide synthesis aretechniques well-known to one skilled in the art, and encompass bothsolid phase and solution phase methods; see generally, Bodanszky andBodanszky, The Practice of Peptide Synthesis, Springer-Verlag, Berlin,1984; Atherton and Sheppard, Solid Phase Peptide Synthesis: A PracticalApproach, IRL Press at Oxford University Press Oxford, England, 1989,and Stewart and Young, Solid phase Peptide Synthesis, 2nd edition,Pierce Chemical Company, Rockford, 1984, the entire contents of each ofwhich are incorporated herein by reference. In both solution phase andsolid phase techniques, the choice of the protecting groups must beconsidered, as well as the specific coupling techniques to be utilized.For a detailed discussion of peptide synthesis techniques for solutionphase and solid phase reactions, see, Bioorganic chemistry: Peptides andProteins, Hecht, Oxford University Press, New York: 1998, the entirecontents of which are incorporated herein by reference.

In certain embodiments, the method comprises a solution phase synthesisof an inventive bifunctional polypeptide. Solution phase synthesis, asmentioned above, is a well-known technique for the construction ofpolypeptides. An exemplary solution phase synthesis comprises the stepsof: (1) providing an amino acid protected at the N-terminus with asuitable amino protecting group; (2) providing an amino acid protectedat the C-terminus with a suitable carboxylic acid protecting group; (3)coupling the N-protected amino acid to the C-protected amino acid; (4)deprotecting the product of the coupling reaction; and

(5) repeating steps (3) to (4) until a desired polypeptide is obtained,wherein at least two of the amino acids coupled at any of the abovesteps each comprise at least one terminally unsaturated amino acid sidechain, and at least one α,α-disubstituted amino acid comprises twoterminally unsaturated amino acid side chains. During the course of theabove synthesis, various parameters can be varied, including, but notlimited to placement of amino acids with terminally unsaturated sidechains, stereochemistry of amino acids, terminally unsaturated sidechain length and functionality, and amino acid residues utilized.

In certain embodiments, the method comprises a solid phase synthesis ofan inventive bifunctional polypeptide or portion thereof. Solid phasesynthesis, as mentioned above, is a well-known technique for theconstruction of polypeptides. An exemplary solid phase synthesiscomprises the steps of: (1) providing a resin-bound amino acid; (2)deprotecting the resin bound amino acid; (3) coupling an amino acid tothe deprotected resin-bound amino acid; (4) repeating steps (3) until adesired peptide is obtained, wherein at least two of the amino acidscoupled at any of the above steps each comprise at least one terminallyunsaturated amino acid side chain, and at least one α,α-disubstitutedamino acid comprises two terminally unsaturated amino acid side chains.During the course of the above synthesis, various parameters can bevaried, including, but not limited to placement of amino acids withterminally unsaturated side chains, stereochemistry of amino acids,terminally unsaturated side chain length and functionality, and aminoacid residues utilized.

After a desired polypeptide is synthesized using an appropriatetechnique, the polypeptide is contacted with a specific catalyst topromote “stapling” or “stitching” of the polypeptide. For example, theresin-bound polypeptide may be contacted with a catalyst to promote“stapling” or “stitching,” or may first be cleaved from the resin, andthen contacted with a catalyst to promote “stitching.”

Different amino acids have different propensities for forming differentsecondary structures. For example, methionine (M), alanine (A), leucine(L), glutamate (E), and lysine (K) all have especially high alpha-helixforming propensities. In contrast, proline (P) and glycine (G) arealpha-helix disruptors.

In certain embodiments, the one or more reaction steps further comprisethe use of a coupling reagent. Exemplary coupling reagents include, butare not limited to, benzotriazol-1-yloxy-tris(dimethylamino)-phosphoniumhexafluorophosphate (BOP),benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP), bromo-tris-pyrrolidino phosphonium hexafluorophosphate(PyBroP), 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC),N,N′-carbonyldiimidazole (CDI),3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT),1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxy-7-benzotriazole (HOBt),2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), 0-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TATU),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU),N,N,N′,N′-tetramethyl-O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)uraniumtetrafluoroborate (TDBTU), and O—(N-succinimidyl)-1,1,3,3-tetramethyluranium tetrafluoroborate (TSTU)).

In certain embodiments, the above reaction of step (iv) furthercomprises a suitable base. Suitable bases include, but are not limitedto, potassium carbonate, potassium hydroxide, sodium hydroxide,tetrabutylammonium hydroxide, benzyltrimethylammonium hydroxide,triethylbenzylammonium hydroxide, 1,1,3,3-tetramethylguanidine,1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N-methylmorpholine,diisopropylethylamine (DIPEA), tetramethylethylenediamine (TMEDA),pyridine (Py), 1,4-diazabicyclo[2.2.2]octane (DABCO), N,N-dimethylaminopyridine (DMAP), or triethylamine (NEt₃).

In certain embodiments, one or more reaction steps are carried out in asuitable medium. A suitable medium is a solvent or a solvent mixturethat, in combination with the combined reacting partners and reagents,facilitates the progress of the reaction there between. A suitablesolvent may solubilize one or more of the reaction components, or,alternatively, the suitable solvent may facilitate the suspension of oneor more of the reaction components; see generally, March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, M. B. Smith andJ. March, 5^(th) Edition, John Wiley & Sons, 2001, and ComprehensiveOrganic Transformations, R. C. Larock, 2^(nd) Edition, John Wiley &Sons, 1999, the entire contents of each of which are incorporated hereinby reference. Suitable solvents for include ethers, halogenatedhydrocarbons, aromatic solvents, polar aprotic solvents, or mixturesthereof. In other embodiments, the solvent is diethyl ether, dioxane,tetrahydrofuran (THF), dichloromethane (DCM), dichloroethane (DCE),acetonitrile (ACN), chloroform, toluene, benzene, dimethylformamide(DMF), dimethylacetamide (DMA), dimethylsulfoxide (DMSO), N-methylpyrrolidinone (NMP), or mixtures thereof.

In other embodiments, one or more reaction steps are conducted atsuitable temperature, such as between about 0° C. and about 100° C.

In certain embodiments, one or more reaction steps involve a catalyst.One of ordinary skill in the art will realize that a variety ofcatalysts can be utilized. Selection of a particular catalyst will varywith the reaction conditions utilized and the functional groups presentin the particular peptide. In certain embodiments, the catalyst is aring closing metathesis (RCM) catalyst. In certain embodiments, the RCMcatalyst is a tungsten (W), molybdenum (Mo), or ruthenium (Ru) catalyst.In certain embodiments, the RCM catalyst is a ruthenium catalyst.Suitable RCM catalysts are described in see Grubbs et al., Acc. Chem.Res. 1995, 28, 446-452; U.S. Pat. No. 5,811,515; Schrock et al.,Organometallics (1982) 1 1645; Gallivan et al., Tetrahedron Letters(2005) 46:2577-2580; Furstner et al., J. Am. Chem. Soc. (1999) 121:9453;and Chem. Eur. J. (2001) 7:5299; the entire contents of each of whichare incorporated herein by reference.

In certain embodiments, the RCM catalyst is a Schrock catalyst, a Grubbscatalyst, a Grubbs-Hoveyda catalyst, a Blechart Catalyst; a Neolyst™ Ml;or a Furstner catalyst.

It will also be appreciated, that in addition to RCM catalysts, otherreagents capable of promoting carbon-carbon bond formation can also beutilized. For example, other reactions that can be utilized, include,but are not limited to palladium coupling reactions, transition metalcatalyzed cross coupling reactions, pinacol couplings (terminalaldehydes), hydrozirconation (terminal alkynes), nucleophilic additionreactions, and NHK (Nozaki-Hiyama-Kishi (Furstner et al., J. Am. Chem.Soc. 1996, 118, 12349)) coupling reactions. Thus, the appropriatereactive moieties are first incorporated into desired amino acids orunnatural amino acids, and then the peptide is subjected to reactionconditions to effect “stapling” or “stitching” and subsequentstabilization of a desired secondary structure.

In another aspect, the present invention provides a method ofsynthesizing an inventive polypeptide comprising the steps of: (1)providing a selected number of amino acids comprising (i) at least twoamino acids, each comprising at least one terminally unsaturated aminoacid side chain, and (ii) at least one α,α-disubstituted amino acidcomprising two terminally unsaturated amino acid side chains; (2)coupling the selected number of amino acids together to generate a firstpeptide; and (3) treating the first peptide with a suitable catalyst toprovide a stapled or stitched peptide.

In certain embodiments, divinyl amino acid as “an α,α-disubstitutedamino acid comprising two terminally unsaturated amino acid side chains”is specifically excluded.

In certain embodiments, each terminally unsaturated amino acid sidechain is reactive toward ring closing metathesis. In certainembodiments, the suitable catalyst is a ring metathesis catalyst. Incertain embodiments, the ring closing metathesis catalyst may generateat least two cross-linked rings by the above method. Depending upon thenature of the selected amino acids and their specific location in thepeptide chain, stitched peptides of the present invention may compriseat least 2, 3, 4, 5, 6, or 7, cross-links, and may comprise one or moreconstitutional/structural isomers (i.e., compounds with the samemolecular weight but having different connectivity).

In certain embodiments, the synthetic method generates one stitchedproduct as a preferred product. As used herein a “preferred product”refers to one constitutional isomer present as the major constituent ina mixture of isomers. In certain embodiments, a “preferred product”refers to one constitutional isomer present as a component in at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, ofan isomeric mixture.

The synthetic method may be further modified to include at least threecross-linking staples by: (1) providing a selected number of natural orunnatural amino acids, wherein said number comprises: (i) at least fouramino acids, each comprising at least one terminally unsaturated aminoacid side chain, and (ii) at least one α,α-disubstituted amino acidcomprising two terminally unsaturated amino acid side chains; (2)coupling the selected number of amino acids together to generate a firstpeptide; and (3) treating the first peptide with a suitable catalyst.

Additionally, the synthetic method may be modified to include at leastthree cross-linking staples by: (1) providing a selected number ofnatural or unnatural amino acids, wherein said number comprises: (i) atleast two amino acids, each comprising at least one terminallyunsaturated amino acid side chain, and (ii) at least twoα,α-disubstituted amino acids, each comprising two terminallyunsaturated amino acid side chains; (2) coupling the selected number ofamino acids together to generate a first peptide; and (3) treating thefirst peptide with a suitable catalyst.

The present invention contemplates any and all types of modifications inorder to provide at least 2, 3, 4, 5, 6, or 7, cross-linked staples intothe polypeptides of the invention.

The above amino acids comprising one to two terminally unsaturated aminoacid side chains are so incorporated into the polypeptide chain in orderto provide proximal terminally unsaturated side chains. These proximalterminally unsaturated side chains may be in the same plane as, or sameside of the polypeptide chain as, each other in any given conformationof the polypeptide. Upon treatment with a suitable catalyst, theseproximal side chains react with each other via “stapling” to provide astitched, conformationally stabilized, polypeptide. In certainembodiments, the proximal terminally unsaturated side chains arearranged such that the resulting “staple” does not interfere with thebiological/therapeutic activity of the stitched polypeptide.

Additional Synthetic Modifications

After “stitching” of the polypeptide, as described above, the method mayfurther comprise additional synthetic modification(s). Any chemical orbiological modification may be made. In certain embodiments, suchmodifications include reduction, oxidation, and nucleophilic orelectrophilic additions to a functional group (e.g., a double bondprovided from a metathesis reaction) of the cross-link to provide asynthetically modified stitched polypeptide. Other modifications mayinclude conjugation of a stitched polypeptide, or a syntheticallymodified stitched polypeptide, with a biologically active agent, labelor diagnostic agent anywhere on the stitched polypeptide scaffold, e.g.,such as at the N-terminus of the stitched polypeptide, the C-terminus ofthe stitched polypeptide, on an amino acid side chain of the stitchedpolypeptide, or at one or more modified or unmodified stitched sites(i.e., to a staple). Such modification may be useful in delivery of thepeptide or biologically active agent to a cell, tissue, or organ. Suchmodifications may allow for targeting to a particular type of cell ortissue.

Thus, in certain embodiments, the above synthetic method furthercomprises: (vii) treating the polypeptide with a suitably reactive agentunder suitable conditions to provide a synthetically modified stitchedpolypeptide.

One of ordinary skill in the art will appreciate that a wide variety ofreactions, conditions, and “suitably reactive agent(s)” may be employedto promote such a transformation, therefore, a wide variety ofreactions, conditions, and reactive agents are envisioned; seegenerally, March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, M. B. Smith and J. March, 5^(th) Edition, John Wiley &Sons, 2001; Advanced Organic Chemistry, Part B: Reactions and Synthesis,Carey and Sundberg, 3^(rd) Edition, Plenum Press, New York, 1993; andComprehensive Organic Transformations, R. C. Larock, 2^(nd) Edition,John Wiley & Sons, 1999, the entirety of each of which is herebyincorporated herein by reference. Exemplary “suitably reactive agents”may be any agent reactive with a multiple bond (e.g., a double or triplebond). In certain embodiments, suitably reactive agents are able toreact with a double bond or triple bond, for example, via ahydrogenation, osmylation, hydroxylation (mono- or di-), amination,halogenation, cycloaddition (e.g., cyclopropanation, aziridination,epoxidation), oxy-mercuration, and/or a hydroboronation reaction, toprovide a functionalized single bond or double bond. As one of ordinaryskill in the art will clearly recognize, these above-describedtransformations will introduce functionalities compatible with theparticular stabilized structures and the desired biologicalinteractions; such functionalities include, but are not limited to,hydrogen, cyclic or acyclic, branched or unbranched, substituted orunsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; substituted or unsubstituted hydroxyl;substituted or unsubstituted amino; substituted or unsubstituted thiol,halo; cyano; nitro; azido; imino; oxo; and thiooxo.

In another aspect, in certain embodiments, the method further comprisestreating the polypeptide with a suitably reactive agent to provide asynthetically modified stitched polypeptide, and treating the modifiedstitched polypeptide with a biologically active agent to provide amodified stitched polypeptide conjugated to a biologically-active agent.

In another aspect, in certain embodiments, the above method furthercomprises treating the polypeptide with a suitable reagent to provide asynthetically modified stitched polypeptide, and treating the modifiedstitched polypeptide with a diagnostic agent to provide a modifiedstitched polypeptide conjugated to a diagnostic agent.

Conjugation of an agent (e.g., a label, a diagnostic agent, abiologically active agent) to the inventive polypeptide may be achievedin a variety of different ways. The agent may be covalently conjugated,directly or indirectly, to the polypeptide at the site of stapling, orto the N-terminus or the C-terminus of the polypeptide chain.Alternatively, the agent may be noncovalently conjugated, directly orindirectly, to the polypeptide at the site of stapling, or to theN-terminus or the C-terminus of the polypeptide chain. Indirect covalentconjugation is by means of one or more covalent bonds. Indirectnoncovalent conjugation is by means of one or more noncovalent bonds.Conjugation may also be via a combination of non-covalent and covalentforces/bonds. The agent may also be conjugated through a covalent ornoncovalent linking group.

Any suitable bond may be used in the conjugation of a biologicallyactive agent and/or diagnostic agent to the inventive polypeptidepresent invention. Such bonds include amide linkages, ester linkages,disulfide linkages, carbon-carbon bonds, carbamate, carbonate, urea,hydrazide, and the like. In some embodiments, the bond is cleavableunder physiological conditions (e.g., enzymatically cleavable, cleavablewith a high or low pH, with heat, light, ultrasound, x-ray, etc.).However, in some embodiments, the bond is not cleavable.

Combinatorial Synthesis of Novel Stapled or Stitched Polypeptides

It will also be appreciated by one of ordinary skill in the art that thesynthetic methods as described above can also be applied tocombinatorial synthesis of stapled or stitched polypeptides. Althoughcombinatorial synthesis techniques can be applied in solution, it ismore typical that combinatorial techniques are performed on the solidphase using split-and-pool techniques. During the course of thecombinatorial synthesis, various parameters can be varied, including,but not limited to, placement of amino acids with terminally unsaturatedside chains, stereochemistry of amino acids, terminally unsaturated sidechain length and functionality, and amino acid residues utilized.

The present invention, in one aspect, provides methods for the synthesisof libraries of stapled or stitched polypeptides, as described above,comprising (1) providing a collection of resin-bound amino acids; (2)deprotecting each of said resin bound amino acids;

(3) separating said collection of deprotected resin bound amino acidsinto n equal portions, wherein n represents the number of differenttypes of amino acids to be coupled; (4) coupling of each of n types ofamino acids to the deprotected amino acid; (5) combining each of the nportions together; and (6) repeating steps (2)-(5) until a desiredpolypeptide is obtained, wherein at least two of the amino acids coupledat any of the above steps each comprise at least one terminallyunsaturated amino acid side chain, and at least one α,α-disubstitutedamino acid comprises two terminally unsaturated amino acid side chains.After a desired polypeptide is synthesized, the resin-bound polypeptidemay be contacted with a catalyst to promote “stitching,” or may first becleaved from the resin, and then contacted with a catalyst to promote“stitching.”

It will be appreciated by one of ordinary skill in the art that thelibraries of compounds having stabilized secondary structures can befurther diversified at specific functional moieties after the desiredstabilized structures are formed. For example, free or latent amino acidfunctionalities may be diversified, or alternatively or additionally,free or latent functionality present on the cross-linkers may bediversified. In particularly preferred embodiments, in but one example,the hydrophilicity of stabilized structures may be increased by theintroduction of hydroxyl moieties. As one of ordinary skill in the artwill realize, the diversification reactions will be selected tointroduce functionalities compatible with the particular stabilizedstructures and the desired biological interactions, and thesefunctionalities include, but are not limited to hydrogen, cyclic oracyclic, branched or unbranched, substituted or unsubstituted aliphatic;cyclic or acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; substituted or unsubstituted acyl; substitutedor unsubstituted hydroxyl; substituted or unsubstituted amino;substituted or unsubstituted thiol; halo; cyano; nitro; azido; imino;oxo; and thiooxo.

The targeting and effector domains A and E of the bifunctional stapledpeptides of the invention can be designed by any method known in theart. For example, A and B can be designed according to known binding orinteraction domains from the literature. Many interaction domains forwell characterized viral and cellular oncogenes (e.g., c-Myc, Ras),tumor suppressors (e.g., p53, Rb), transcription factors (e.g., HIF,E2F), modifying enzymes (e.g., ubiquitin ligases, acetylases,phosphorylases, methylases), structural proteins, signaling receptorsand signaling pathway molecules (e.g., beta-catenin), growth factors(e.g., EGFR) are known in the art.

Effector domains can be designed, for example, for binding to andrecruitment of co-repressor proteins such as Groucho/TLET, SHARP, NCoR,NCoR2, SMRT, BCoR or others. For example, the engrailed homology (Ehl)domains that are found in transcription factors and are known to beessential and sufficient for recruiting Groucho/TLE1 co-repressors totarget promoters may serve to design the effector domain.

In another embodiment, the amphipathic alpha-helix of Mad1 that bindsand retains the Sin-3 repressive complex through its PAH domain may beused to design the effector domain. In another embodiment, the effectordomain (E) is designed according to the FXXFF motif capable of bindingand recruiting MDM2 or MDMX or according to the p53 activation domain 1(Ac-LSQETFSDLWKLLPE-CONH2 (SEQ ID NO:35)).

In another embodiment, the effector domain (E) is designed to bind andrecruit molecules belonging to the the nuclear export machinery, such asexportins, e.g. CRM1.

In another embodiment, the effector domain (E) is designed as a signalpeptide or small molecule comprising or mimicking a nuclear localizationsequence (NLS) to bind nuclear import proteins, e.g. NLS sequences thatare known to target and bind Impα. Examples of NLS sequences are SV40T-antigen: Ac-PKKKRKVE-CONH2 (SEQ ID NO:42); Nucleoplasmin:Ac-KRPAATKKAGQAKKKKLD-CONH2 (SEQ ID NO:43); and c-Myc:Ac-PAAKRVKLD-CONH2 (SEQ ID NO:44) (Gorlich D and Kutay U. Annu. Rev.Cell Dev. Biol. 1999, 15: 607-60).

In another embodiment, the effector domain (E) is designed as a peptideor small molecule capable of binding and recruiting specifictranscriptional co-activator proteins or components of the basaltranscriptional apparatus, for example, TAFII proteins or RNApolymerases or an effector domain according to the KIX domain ofCBP/p300 that has two distinct binding sites targeted by transcriptionfactors to localize and retain the co-activator protein. These domainsmay include p53 AD1: Ac-LSQETFSDLWKLLPE-CONH₂ (SEQ ID NO:45); p53 AD2:Ac-MLSPDDIEQWFTEDPG-CONH₂ (SEQ ID NO:46); MLL: Ac-ILPSDIMDFVLKNTP-CONH₂(SEQ ID NO:47); c-Jun: Ac-LASPELERLIIQSSN-CONH₂ (SEQ ID NO:48);HLTV-TAX: Ac-YIDGFVIGSALQFLIPRLP-CONH₂ (SEQ ID NO:49); c-MYB:Ac-KEKRIKELELLLMSTENELKG-CONH₂ (SEQ ID NO:50); pKID:Ac-ILSRRPSYRKILNDLSSDAPG-CONH₂ (SEQ ID NO:51), or derived from p-KID,where any serine residues, in particular Ser133, can be foundphosphorylated, as is present in the native pKID:KIX interaction.

In another embodiment, the effector domain (E) is designed comprisingpeptides or small molecules capable of binding and recruiting specificpost-translational modifying enzymes or complexes including kinases,acetyltransferases, phosphatases, glycotransferases, lipid transferasesand others to alter transcription factor function.

In another embodiment, the targeting domain is designed according to atranscription factor targeting ligand, such as SAHM1, capable of bindingthe Notch:CSL transcription factor complex.

High affinity targeting and effector domains (A and E) can also bedesigned rationally according to available crystallographic data or dataderived from published affinity screens, such as phage display.

The targeting and effector domains A and E of the bifunctional stapledpeptides of the invention can be obtained for any protein that isdesired using, for example, high throughput affinity screens. Forexample, the targeting domains can be designed to associate or bind toany candidate oncogene, tumor suppressor, transcription factor, such asthe NF-kappaB and AP-1 families of transcription factors, the STATfamily members, the steroids receptors, Ets factors, ATF family members,basic helix-loop-helix transcription factors, telomerases, growthfactors, and growth factor receptors. These factors might be, forexample, misfunctional, mislocalized, deregulated, ectopically active,inactive, or misfolded and may contribute to cellular transformation,changes in cell fate, de-differentiation, apoptosis, necrosis, ectopiccell signaling or other changes that cause a disease state in a subject.These candidate proteins associated with one targeting domain of thebifunctional stapled peptides of the invention can then be tethered toan effector biomolecule, as described herein, such as ubiquitin ligases,DUBs, acetylases, deacetylases, kinases phosphatases, methylases,demethylases, glycosyltransferases, glycosidases, or chaperones. Thetargeting and effector domains can, in certain embodiments, besubstantially similar to or homologous with a known bioactivepolypeptide, e.g., a polypeptide that is known to bind or associate witha target biomolecule or effector biomolecule.

In certain embodiments, the targeting and effector domains A and Ecomprising one or more non-natural amino acids and/or one or morecross-linking moieties are selected for high binding affinity andbinding specificity as well as activity using any high throughputaffinity screens know in the art, such as phage display, or as describedin FIGS. 15 and 16.

In certain embodiments, the targeting and effector domains A and E arecovalently associated by a linker L. This linker can have any length orother characteristic and minimally comprises two reactive terminalgroups that can chemically interact with (and covalently bind to) thepolypeptide chains of targeting and effector domains A and E.

In some embodiments, the linker L can comprise natural or non-naturalamino acids and/or may comprise other molecules with terminal reactivegroups. For example, linkers may comprise PEG (polyethylene glycol) andNHS or maleimide reactive terminal groups, such as, SM(PEG)_(n)Succinimidyl-([N-maleimidopropionamido]-n-ethyleneglycol). It will beappreciated that the length of the linker L is variable and can bedesigned based on the required flexibility or rigidity necessary to linkthe targeting and effector domains A and E, respectively. Examples ofcovalent conjugation strategies and suitable reaction conditions using avariety of linkers and/or functional groups to associate polypeptide Awith polypeptide E are set forth in FIGS. 27 to 32. In certainembodiments, thiol-maleimide conjugates are generated. In otherembodiments, 1,4- or 1,5-triazole conjugates are generated from thereaction of an azide with an alkyne.

The distance of the targeting and effector domains A and E can be variedby the length of linker L based on several parameters, including, butnot limited to: 1) the molecular size of the respective targetbiomolecule and effector biomolecule, and/or 2) the relative moleculardistance between the catalytic site of the effector biomolecule and thesite of modification on the target biomolecule, and/or 3) the relativedistance of the sites on the effector biomolecule and the targetbiomolecule that are bound by the targeting domains A and E, and/or 4)cell permeability of the bifunctional stapled peptide, and/or 5)bioavailability of the bifunctional stapled peptide, and/or 6) stabilityof the bifunctional stapled peptide, and/or other structural or chemicalconsiderations.

EXAMPLES Example 1: Bifunctional Stapled Peptide for Degradation ofβ-Catenin

β-catenin is an essential component of the Wnt signaling pathway. Thecanonical Wnt pathway plays critical roles in embryonic development,stem cell growth, and tumorigenesis. Stimulation of the Wnt pathwayleads to the association of β-catenin with Tcf and BCL9 in the nucleus,resulting in the transactivation of Wnt target genes. The level ofβ-catenin in the cytosol is regulated by β-catenin destruction complex.In the absence of a Wnt signal, β-catenin is phosphorylated, leading toits ubiquitination by the SCF E3 ubiquitin ligase complex and subsequentdegradation by the 26S proteasome (FIG. 1). In the presence of a Wntsignal, the destruction complex is inhibited and cytosolic levels ofβ-catenin rise, allowing its translocation to the nucleus whereβ-catenin interacts with Tcf and other transcription factors to activatetarget genes. Genetic aberrations in components of this pathway areassociated with a variety of cancers [Barker and Clevers, NRDD, 5,998-1014 (2006)]. Because most colon cancers are caused by an excessaccumulation of β-catenin, the Wnt signaling pathway might be a goodtarget for the development of Wnt signaling inhibitors for cancertreatment. The molecular structure of β-catenin has been resolved[Sampietro et al., Mol. Cell 24, 293-300 (2006)]. The sites ofinteraction of β-catenin with the transcriptional co-activator Bcl9 andthe DNA-binding transactivator Tcf-4 have been identified and resolvedas a triple complex in a crystal structure [Sampietro et al., Mol. Cell24, 293-300 (2006)]. For example, the BCL9 β-catenin binding domain(CBD) forms an α helix that binds to the first armadillo repeat ofβ-catenin. In many cancers, mutations of proteins involved in β-cateninubiquitination and degradation are frequently found, resulting inaberrant levels of β-catenin (FIG. 2). For example, most colorectalcancers have mutations of the adenomatous polyposis coli (APC) gene orthe beta-catenin gene that stabilize beta-catenin and activatebeta-catenin target genes leading to cancer.

Bifunctional stapled peptides are used to restore β-catenin destructionthrough polyubiquitination in cancer cells that harbor mutations in theβ-catenin destruction pathway (for example mutated or truncatedadenomatous polyposis coli, APC). The bifunctional stapled peptides havetwo moieties, a targeting and an effector domain. The targeting domainis a β-catenin binding moiety and the effector domain is a E3 ubiquitinligase recruiting moiety. The E3 ubiquitin ligase binding moietyrecruits E3 ubiquitin ligase in proximity to (3-catenin and therebyfacilitates β-catenin polyubiquitination (FIG. 3). There are severalsurface exposed lysine residues in the Arm-domain of β-catenin (FIG. 4).

Suitable bifunctional stapled peptides are transfected in vitro intocancer cells, for example colon cancer cells (SW480, DLD-1, and HT29,HCT-116), breast cancer cells (MCF7), or prostate cancer cells (PC3,LNCAP), and transfected cells are screened by western blot analysis fora reduction of soluble and/or membrane-bound (cytosolic and/or nuclear)β-catenin protein levels or appearance of poly-ubiquitinated forms ofβ-catenin, and using reporter assays (e.g., luciferase) to detectdown-regulation of co-transfected LEF/TCF target genes. Cellulardistribution of β-catenin (nuclear, cytosolic) is followed byimmunofluorescence, using β-catenin specific antibodies and standardstaining protocols. As a positive control, CELECOXIB is used.

Several bifunctional stapled peptides are tested for their ability torestore polyubiquitination of β-catenin: Bcl 9-SAH p53-8 and Tcf4-SAHp53. hDM2 is a E3 ligase well known to promote p53 degradation viaubiquitination. A stapled peptide SAHp53 was previously synthesized as adominant negative that binds to hDM2. The SAHp53-8 is used as the E3ligase recruiting moiety (effector domain) to bring hDM2 in proximity toβ-catenin. Bcl-9 and Tcf4 peptides possess α-helical structure that canbe stapled. The Bcl-9 or Tcf4 peptides are fused with SAH p53-8. Theresulting bifunctional peptide bridges β-catenin and hDM2 and therebyfacilitates β-catenin ubiquitination (FIGS. 5 and 6).

Bifunctional stapled peptides with different orientations are produced.In the design of bifunctional stapled peptides, SAH p53-8 is placed ateither N- or the C-terminus (FIG. 7). Peptides are then screened toselect the optimal orientation. The following bifunctional peptides aresynthesized:

Group 1: SAH p53-8-Bcl 9 (SEQ ID NO: 1)QSQQTFR₈NLWRLLS₅QN-(Ahx)_(n)-SQEQLR₈HRERSLS₅TLRDIQRMLF (SEQ ID NO: 2)QSQQTFR₈NLWRLLS₅QN-(Ahx)_(n)-SQEQLEHRERSLS₅TLRS₅IQRMLF (SEQ ID NO: 3)QSQQTFR₈NLWRLLS₅QN-(Ahx)_(n)-SQEQLEHRS₅RSLS₅TLRDIQRMLFn = 4, Ahx: aminohexanoic acid Group 2: Bcl 9-SAH p53-8 (SEQ ID NO: 4)SQEQLR₈HRERSLS₅TLRDIQRMLF-(Ahx)_(n)-QSQQTFR₈NLWRLLS₅QN (SEQ ID NO: 5)SQEQLEHRERSLS₅TLRS₅IQRMLF-(Ahx)_(n)-QSQQTFR₈NLWRLLS₅QN (SEQ ID NO: 6)SQEQLEHRS₅RSLS₅TLRDIQRMLF-(Ahx)_(n)-QSQQTFR₈NLWRLLS₅QNn = 4, Ahx: aminohexanoic acid Group 3: SAH p53-8-Bcl 9 (SEQ ID NO: 7)QSQQTFR₈NLWRLLS₅QN-(PEG)_(n)-SQEQLR₈HRERSLS₅TLRDIQRMLF (SEQ ID NO: 8)QSQQTFR₈NLWRLLS₅QN-(PEG)_(n)-SQEQLEHRERSLS₅TLRS₅IQRMLF (SEQ ID NO: 9)QSQQTFR₈NLWRLLS₅QN-(PEG)_(n)-SQEQLEHRS₅RSLS₅TLRDIQRMLFn = 4, PEG: Polyethyleneglycol Group 4: Bcl 9-SAH p53-8 (SEQ ID NO: 10)SQEQLR₈HRERSLS₅TLRDIQRMLF-(PEG)_(n)-QSQQTFR₈NLWRLLS₅QN (SEQ ID NO: 11)SQEQLEHRERSLS₅TLRS₅IQRMLF-(PEG)_(n)-QSQQTFR₈NLWRLLS₅QN (SEQ ID NO: 12)SQEQLEHRS₅RSLS₅TLRDIQRMLF-(PEG)_(n)-QSQQTFR₈NLWRLLS₅QNn = 4, PEG: Polyethyleneglycol

Aminohexanoic acid (Ahx) or polyethyleneglycol (PEG) ranging from 2-4residues is used as a linker to connect the two stapled peptides (FIGS.8 and 9). The optimal length of the Ahx linker is determined empiricallybased on biochemical as well as cell based assays.

Group 5: SAH p53-8-Tcf-4 (SEQ ID NO: 13)QSQQTFR₈NLWRLLS₅QN-(Ahx)_(n)-DELISFKDEGEQE(β-Ala)₂ ERD LS₅DVKS₅SLVN(SEQ ID NO: 14) QSQQTFR₈NLWRLLS₅QN-(Ahx)_(n)-DELISFKDEGEQE(β-Ala)₂ ER₈DLADVKS₅SLVN n = 2-4, Ahx: aminohexanoic acid, β-Ala: β-AlanineGroup 6: Tcf-4-SAH p53-8 (SEQ ID NO: 15)DELISFKDEGEQE(β-Ala)₂ ERDLS₅DVKS₅SLVN-(Ahx)_(n)-QSQQTF R₈NLWRLLS₅QN(SEQ ID NO: 16) DELISFKDEGEQE(β-Ala)₂ ER₈DLADVKS₅SLVN-(Ahx)_(n)-QSQQTFR₈NLWRLLS₅QN n = 2-4, Ahx: aminohexanoic acid, β-Ala: β-AlanineGroup 7: SAH p53-8-Tcf-4 (SEQ ID NO: 17)QSQQTFR₈NLWRLLS₅QN-(PEG)_(n)-DELISFKDEGEQE(β-Ala)₂ ERD LS₅DVKS₅SLVN(SEQ ID NO: 18) QSQQTFR₈NLWRLLS₅QN-(PEG)_(n)-DELISFKDEGEQE(β-Ala)₂ ER₈DLADVKS₅SLVN Group 8: Tcf-4-SAH p53-8 (SEQ ID NO: 19)DELISFKDEGEQE(β-Ala)₂ ERDLS₅DVKS₅SLVN-(PEG)_(n)-QSQQTF R₈NLWRLLS₅QN(SEQ ID NO: 20) DELISFKDEGEQE(β-Ala)₂ ER₈DLADVKS₅SLVN-(PEG)_(n)-QSQQTFR₈NLWRLLS₅QN

Aminohexanoic acid (Ahx) or polyethyleneglycol (PEG) ranging from 2-4residues is used as a linker to connect the two stapled peptides (FIGS.10 and 11). The optimal length of the Ahx linker is determinedempirically based on biochemical as well as cell based assays.

Heterofunctional crosslinkers are used to join the two peptide domainstogether. The NHS ester attacks the primary amine on peptide 1 to forman amide bond, and the maleimide group reacts to free thiol groups suchas cysteines on peptide 2 (FIG. 12). FIG. 13 shows typical spacersbetween NHS and Maleimide, ranging from 2-24 units. The advantage ofusing this type of crosslinker is that the crosslinked product peptidedoes not have to be in a specific orientation since the cysteines can beplaced at either end of peptide 2 (if peptide 1 is already reacted).FIG. 14 shows that with the NHS-maleimide crosslinker, the twofunctional peptides can be joined either in the orientation of N to C orN to N as long as there is a cysteines incorporated in the peptideeither at the N-terminal or C-terminal end. This results in segmentcross-linking in two orientations:

Orientation 1: (SEQ ID NO: 21)SAH p53-8-CDELISFKDEGEQE(β-Ala)₂ ERDLS5DVKS5SLVN (SEQ ID NO: 22)SAH p53-8-CDELISFKDEGEQE(β-Ala)₂ ER8DLADVKS5SLVN Orientation 2:(SEQ ID NO: 23) SAH p53-8-DELISFKDEGEQE(β-Ala)₂ ERDLS5DVKS5SLVNC(SEQ ID NO: 24) SAH p53-8-DELISFKDEGEQE(β-Ala)₂ ER8DLADVKS5SLVNC

Example 2: Screening Procedures to Obtain High Affinity Targeting andEffector Domains

Bifunctional stapled peptides are screened for high affinity bindingusing various approaches:

1) Synthetic libraries of stapled peptides: The purpose of such ascreening is the identification of stapled peptide sequences capable ofbinding to a specific protein. Libraries are constructed by split-poolsynthesis. The peptide sequences is synthesized on bead (split and pool)and is composed of a constant subunit (such as p53, TCF4, or Axinderived stapled peptides) and of a variable subunit (FIG. 15). Thevariable subunit can be designed based on i,i+4 and i,i+7 architecture(X=random amino acid), e.g.:

i,i+4: XXX—S₅—XXS—S₅—XX  1

i,i+7: XXX—R₈—XXSSXX—S₅  2

A combinatorial library analogue to sequence 2 was assembled. For X areduced set of 10 amino acids was chosen (R, Q, F, L, A, W, V, S, H, Y).The assembled sequences were determined by Edman degradation.Dye-labeled target proteins are screened for their ability to interactwith the beads (FIG. 15). Sequences of hits are read out by Edmansequencing. In another approach (FIG. 16), a constant region is used tomediate binding to an enzyme (e.g., p53-MDM2 interaction). Due to theenzymatic activity a second protein bound to the variable sequence canbe modified. In a subsequent step the induced modification is detectedand used as selection criterion.

2) Phage Display: A template helical peptide, APP, is expressed on thepIII coat protein of M13 phage. At least 10 positions are randomized,using all codons encoding all 20 naturally occurring amino acids.Positives are identified by panning. Sequences are optimized byerror-prone PCR. High-affinity hits are confirmed using syntheticpeptides.

3) Yeast Cell Surface Display: The procedure is carried out as outlinedin 2), however the APP is expressed on the outside surface ofSaccharomyces cerevisiae.

Example 3: Axin-Derived Stapled α-Helices for Use in BifunctionalPeptides

Additional variant bifunctional peptides are synthesized:

(SEQ ID NO: 23) SAH p53-8-DELISFKDEGEQE(β-Ala)₂ ERDLS₅DVKS₅SLVNCTcf-4: Kd˜100 nM, as in Example 1. Axin-derived stapled α-helices areused (ENPES ILDEHVQRVMR, SEQ ID NO: 25, Kd˜3 μM).

(SEQ ID NO: 26) SAH p53-8-NPE-S₅-ILD-S₅-HVQRVMR (SEQ ID NO: 27)SAH p53-8-NPESILD-S₅-HVQ-S₅-VMR (SEQ ID NO: 28)SAH p53-8-NPE-R₈-ILDEHV-R₅-RVMR

Affinity is increased as compared to the non-stapled wild type sequenceSEQ ID NO: 25 (Kd˜3 μM). In addition these shorter all-helical peptidesexhibit higher cell permeability than the TCF4 derived sequences, whichconsist of a helical and an unstructured subunit.

Example 4: Bifunctional Stapled Peptides for Degradation of c-Myc

c-Myc is a master regulator of genes involved in cell growth, proteinsynthesis and metabolism and a key positive cell cycle regulator. It isinappropriately activated in ca. 30% of all human tumors and as such isconsidered, after K-Ras, to be the second most frequently activatedoncoprotein in human cancer [see Nat. Rev. Mol. Cell Biol. 9, 810-5(2008); Nature 455, 679-83 (2008); Nat. Rev. Mol. Cell Biol. 6, 635-45(2005); Nat. Rev. Mol. Cell Biol. 5, 805-10]. Structurally, c-Myc is amember of the basic helix-loop-helix leucine zipper (bHLH-Zip)transcription factor family. C-Myc is itself a momoneric protein, butits ability to regulate gene expression is dependent upon formation of aDNA-binding heterodimer with partner proteins of the bHLH-Zip family,namely Mad, Max, and Mxi-1. The structure of the c-Myc is known [Nairand Burley, Cell 112, 193-205 (2003)]. Max specifically dimerizerizeswith c-Myc, and c-Myc/Max heterodimers function as transcriptionalactivators, binding the E-box hexanucleotide motif. Mad, and Mxi-1 areantagonizing the cell cycle promoting activity of the c-Myc/Maxheterodimers. Mad and Mxi can heterodimerize with Max, depriving c-Mycof a partner. The Max/Mad or Max/Mxi-1 partners either fail to activateor actively repress transcription, leading to a state of growthinhibition, quiescence, and/or cell differentiation. Max proteins aremetabolically stable and are constitutively expressed, while c-Myc, Mad,and Mxi-1 are unstable, responding to the level of mitotic stimulationin the cell.

c-Myc activity and stability are regulated by phosphorylation andubiquitination. For example, increased phosphorylation of c-Myc at Thr58can induce degradation of c-Myc via ubiquitination-proteasomaldegradation. The E3 ligase complex responsible for the degradation ofc-Myc is a SCF complex associated with the F-box protein FBW7. Fbxw7(also known as Fbw7, Sel-10, hCdc4, or hAgo) induces the degradation ofpositive regulators of the cell cycle, such as c-Myc, c-Jun, cyclin E,and Notch. FBXW7 is often mutated in a subset of human cancers.

Increased levels of c-myc, for example as a result of reduceddegradation via proteasome can lead to cancer. However, ectopicallyactive SCF (for example by overexpression of Skp2) can also contributeto cancer, because SCF also targets p27^(KIP1) for proteasomaldegradation. p27^(KIP1) is an inhibitor of cyclin-dependent kinases(e.g., CDK1 and CDK2) and an important negative regulator of the cellcycle. Degradation of p27 CKI leads to increase tumor aggressiveness andworsening of prognosis in several types of human cancers.

A main advantage of specifically targeting SCF or another E3 ligase toc-Myc using the bifunctional peptides described herein is that c-Mycdegradation can be specifically induced without simultaneously inducingthe degradation of other factors, such as, for example, p27^(KIP1).

Bifunctional peptides comprising a targeting domain and an effectordomain are synthesized that tether c-Myc and an E3 ligase to promote thedegradation of c-Myc. In addition, bifunctional peptides comprising atargeting domain and an effector domain are synthesized that tetherc-Myc and a kinase increasing c-Myc phosphorylation, for example, thephosphorylation of Thr58 to promote the degradation of c-Myc. Inaddition, bifunctional peptides comprising a targeting domain and aneffector domain are synthesized that tether Max constitutively (thatmeans independent of mitogenic stimuli) to either Mad or Mxi-1 todeprive c-Myc of its activating partner and to inactivate c-Myc.

Example 5: Bifunctional Stapled Peptides for Degradation of HIF

Hypoxia-inducible factor (HIF) is a transcriptional regulatory proteinthat controls genes involved in angiogenesis, glucose utilization, andresistance to hypoxic stress. HIF is believed to be essential for thegrowth of solid tumors, as escape from hypoxia-induced apoptosis is anecessary precondition for the formation of a tumor mass larger than afew tenths of a millimeter. More recently, it has been appreciated thatHIF also has a profound role in energy utilization, upregulating theexpression of glycolytic genes in cell states during which they wouldordinarily be quiescent. This raises the intriguing possibility that HIFinhibition will be useful in treating both solid and blood-bornecancers. HIF is a heterodimer comprising one unit of an induciblesubunit, HIF-1α, and a constitutive subunit known as ARNT or HIF-1β.Both subunits are members of the basic-helix-loop-helix structuralfamily (bHLH) and so are structurally related to cMyc, but both HIFsubunits lack the leucine zipper motif of c-Myc.

HIFα activity is regulated by enzymatic oxygen-dependent hydroxylationof two specific prolyl residues and one critical asparaginyl residue bythe oxoglutarate-dependent dioxygenases PHD 1-3 and a protein termedfactor inhibiting HIF (FIH). Prolyl hydroxylation results in vonHippel-Lindau (VHL) complex-mediated ubiquitination of HIFα andconsequent degradation by the proteasome. Similarly, asparaginylhydroxylation inhibits CBP/p300 coactivator recruitment by HIFα chains(Bruick & McKnight, 2002). Inactivation of the VHL gene (e.g., bymutation) is associated with the development of highly vascularizedtumors.

Bifunctional peptides comprising a targeting domain and an effectordomain are synthesized that tether HIF-1α or HIF-1β and an E3 ligase topromote the degradation of HIF-1α or HIF-1β.

Example 6: Bifunctional Peptides for Promotion of GTPase Functions ofMutated Ras

Ras is a small GTP binding protein that operates as a molecular switchregulating the control of gene expression, cell growth, anddifferentiation through a pathway from receptors to mitogen-activatedprotein kinases (MAPKs). Oncogenic mutations in the human Ras genes (H-,N-, and K-Ras) are observed in 30% of human cancers. Pancreas, colon,and lung tumors are most often associated with Ras mutations. Mostmutations have been detected in the K-Ras gene, and they typicallyinvolve missense substitutions of the encoded GTPase in one of threeamino acid positions (12, 13, or 61) that occupy the catalytic site ofGTP hydrolysis. The mutated forms of Ras remain GTP-bound, and transduceconstitutive signals for cell proliferation.

The intrinsic catalytic activity of the Ras GTPase is inefficient andrequires a GTPase-activating protein (GAPs) to function as anoff-switch. Four types of Ras-specific GAPs have been identified,including p120 Ras GAP, neurofibromin (NF-1), SynGAP, and the GAP1family. Ras mutational substitutions lead to diminished intrinsic GTPaseactivity, and to resistance to GTPase stimulation by Ras-specific GAPs.

The GTPase defect in oncogenic Ras is based in part on glutamine 61 ofRas that activates a water molecule for nucleophilic attack, and itssubstitution by any other amino acid abolishes both intrinsic andGAP-stimulated GTPase activity. The crystal structure of the Ras-RasGAPcomplex revealed that any mutation of glycine 12 or glycine 13 positionsa side-chain that both displaces glutamine 61 and sterically occludesthe catalytic ‘arginine finger’ of GAP (R789), resulting in a loss ofintrinsic and GAP-stimulated GTPase activity [Scheffzek et al. Science,(1997) 277: 333-338], with the exception of a Ras proline 12 mutant,which activates intrinsic GTPase activity. Ras proline 12 cannottransform cultured cells, suggesting that partial restoration of theGTPase activity of oncogenic Ras mutants might prevent oncogenesis.

Small molecule-based therapies designed to target Ras are currentlybased on inhibition of the enzyme FTase. FTase catalyzes theCOOH-terminal farnesylation of Ras, a post-translational modificationthat is essential for Ras function. However, these inhibitors do notselectively target the oncogenic forms of Ras, and, may disrupt thefunctions of wild-type Ras that are required in normal cells. Fischbachet al. [Cancer Research (2003) 63, 4089-4094] have shown that nucleosidediphosphate kinase (Ndk, human ortholog NM23) is a metastasis suppressoreffectively inactivates several of the oncogenic forms of Ras that areseen frequently in human cancers, including RasD12 and does notdetectably affect wild-type Ras or an activated form of the Ras-relatedRho GTPase.

Bifunctional peptides of the invention are used to tether GAPs and/orNdk to mutated Ras to promote GTPase function.

Example 7: Bifunctional Peptides of Phosphorylation of STAT

In alternative approaches to directly modifying Ras and/or c-Mycdownstream effectors, such as STAT3 and STAT5 can be modified using thebifunctional peptides of the invention. STAT 3 and STAT 5 arephosphorylated and active in many cancers, for example in Ras and/orc-Myc transformed cancers. Inactivation of oncogenic Ras or c-Myc leads,in certain cancers, to de-phosphorylation of STAT 3 and STAT 5 andregression of the cancer. Bifunctional peptides of the invention areused to tether a specific phosphatase to STAT3 and/or STAT 5 todephosphorylate STAT3 and/or STAT 5.

Example 8: Transcription Factor Degradation Through TargetedUbiquitination

The effector domain (E) is designed as a signal peptide or smallmolecule capable of binding and recruiting a ubiquitin-ligase protein,such as MDM2 or FBXW7. The proximity of the ubiquitin-ligase proteinbound to the effector domain (E) and a transcription factor (ortranscription factor complex) bound to the targeting domain (A) leads toenhanced ubiquitination or restored ubiquitination, if, for example,wild-type ubiquitination sites on the target protein have been mutatedand ubiquitination no longer occurs at these sites, for example inβ-catenin, Notch, and c-Myc. Ubiquitination of the transcription factorleads to proteasomal degradation (see FIG. 17). For example, theeffector domain (E) is designed according to the FXXFF motif-containingstapled peptides capable of binding and recruiting MDM2 or MDMX; the p53activation domain 1: Ac-LSQETFSDLWKLLPE-CONH₂ (SEQ ID NO:35), which canbe stapled, and/or may comprise non-natural amino acids; small moleculescapable of binding MDM2, such as Nutlin-3; or peptides capable ofbinding FBXW7 E3-Ubiquitin ligase. The following stapled peptides areuseful as effector domains:

(SEQ ID NO: 36) Ac-LSQETFS*LWK*LPE-CONH₂ (SEQ ID NO: 37)Ac-QSQQTF#NLWRKK*QN-CONH₂ (SEQ ID NO: 38) Ac-QSQQTF*NLW*KKQN-CONH₂(SEQ ID NO: 39) Ac-LSQNTFS*LWK*LPQ-CONH₂

-   -   Where “*” is the non-natural amino acid S5 and “#” is the        non-natural amino acid R8. In any arrangement, these amino acids        are cross-linked.

Any part of the targeting domain A may be linked to any part of theeffector domain E through the linker L. For example, the linkage isN-terminus to N-terminus, the linkage is C-terminus to N-terminus, thelinkage is C-terminus to C-terminus, or the linkage is through interioramino acids of one or both peptides. The linkage is typically positionedin such a way as to avoid interfering with the binding activity of thepeptide and/or to avoid interfering with the stapling of the peptide.The linker can be proteinogenic or non-proteinogenic. The linker can bea covalent bond (e.g., a carbon-carbon bond, disulfide bond,carbon-heteroatom bond), or it can be a polymeric linker (e.g.,polyethylene, polyethylene glycol, polyamide, polyester). The linker cancomprise a monomer, dimer, or polymer of aminoalkanoic acid, or thelinker can comprise an aminoalkanoic acid (e.g., glycine, ethanoic acid,alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid,5-pentanoic acid). For example, the linker can comprise a monomer,dimer, or polymer of aminohexanoic acid (Ahx) or polyethylene glycolmoiety (PEG). The linker can comprise amino acids. The linker caninclude functionalized moieties to facilitate attachment of anucleophile (e.g., thiol, amino) from the peptide to the linker. Thelinker can include a maleimide group or a NHS ester or the linkerincludes both a NHS ester and a maleimide group.

Example 9: Transcription Factor Target Gene Repression ThroughRecruitment of Co-Repressors

The effector domain (E) is designed as a domain capable of binding andrecruiting co-repressors, histone deacetylases (HDACs), or other generaltranscription repressors, imposing active repression at transcriptionfactor target-gene promoters and/or repression through epigeneticchanges, e.g. through HDAC-mediated chromatin condensation (see FIG.18). The effector domain (E) is designed as a signal peptide or smallmolecule capable of binding co-repressor proteins such as Groucho/TLE1,SHARP, NCoR, NCoR2, SMRT, BCoR, or others.

For example, engrailed homology (Ehli) domains that are found intranscription factors and are known to be essential and sufficient forrecruiting Groucho/TLE1 co-repressors to target promoters are designed.This domain relies on a short peptide sequence for the interaction:Ac-TPFYIEDILG-CONH₂ (SEQ ID NO:40). “E” peptides or peptidomimetics ofthis domain tethered to “A” enact target-gene repression.

In another example, the amphipathic alpha-helix of Mad1 that binds andretains the Sin-3 repressive complex through its PAH domain is designed(see FIG. 24A). A natural or stapled variant of this peptide sequenceserves as an effective “E” domain: Ac-VRMNIQMLLEAADYLERRER-CONH₂ (SEQ IDNO:41).

Examples of stapled “E” domains from Mad1:

(SEQ ID NO: 67) Ac-VRMNIQMLLEA*DYL*RRER-CONH₂ (SEQ ID NO: 68)Ac-VRMNIQM*LEA*DYLERRER-CONH₂ (SEQ ID NO: 69)Ac-VRMNIQML#EAADYL*RRER-CONH₂ (SEQ ID NO: 70)Ac-VRM*IQM*LEAADYLERRER-CONH₂

-   -   Where “*” is the non-natural amino acid S5 and “#” is the        non-natural amino acid R8. In any arrangement, these amino acids        are cross-linked.

Example 10: Transcription Factor Inhibition by Targeted Nuclear Exportwith Nuclear Export Sequence (NES)-Containing Bi-Functional Peptides

The effector domain (E) is designed as a domain capable of binding andrecruiting the nuclear export machinery, thus targeting the“A”-transcription factor complex for nuclear export to the cytosol.Active export by exportins such as CRM1 disable the transcriptionfactor-specific gene expression programs by spatially preventingtranscription factor function (see FIG. 19). The effector domain “E” isdesigned as signal peptides or small molecules capable of bindingnuclear export proteins such as CRM1 (Exportin 1). Many CRM1-interactingNES domains have been discovered and usually consist of a 10-20 residuepeptide with a 5-6 residue hydrophobic core. For example, the HR3 domainin the dengue virus NS5 protein, which has been found to export avariety of fused protein cargo is such domain. An “E” fusion of thepeptide: Ac-LLTKPWDIIPMVTQMAM-CONH₂ (SEQ ID NO:71) is made whichpromotes nuclear export of the target transcription factor (Rawlinson SM et al., J.B.C.. 2009, 284, 15589-97). Another example of a short,well-characterized NES that interacts with CRM1 is from the activationdomain of the HIV-1 REV protein. An “E” fusion: Ac-CLRRLERLTL-CONH₂ (SEQID NO:72) has been shown to promote export of fusion proteins andfurthermore a mutant (LE to DL) is inactive, indicating a specificinteraction. (Fischer U et al., Cell, 1995, 82, 475-83).

Example 11: Transcription Factor Activation by Targeted Nuclear Importwith Nuclear Localization Sequence (NLS)-Containing Bi-FunctionalPeptides

The effector domain (E) is designed as signal peptides or smallmolecules comprising or mimicking a nuclear localization sequence (NLS)to bind nuclear import proteins (see FIG. 20). NLS sequences that areknown to target and bind Impα are designed.

Exemplary NLS sequences are:

SV40 T-antigen: (SEQ ID NO: 42) Ac-PKKKRKVE-CONH₂; Nucleoplasmin:(SEQ ID NO: 43) Ac-KRPAATKKAGQAKKKKLD-CONH₂; c-Myc (SEQ ID NO: 44)Ac-PAAKRVKLD-CONH₂.

-   -   (Gorlich D and Kutay U. Annu. Rev. Cell Dev. Biol. 1999, 15:        607-60)

Example 12: Synthetic Transcription Factor Activation by Recruitment ofCo-Activator Proteins

The effector domain (E) is designed as peptides or small moleculescapable of binding and recruiting specific transcriptional co-activatorproteins or components of the basal transcriptional apparatus. Synthetictranscriptional activation enables augmented gene expression driven byspecific transcription factors or a return to basal gene expressionlevels for transcription factors that have been inappropriatelysuppressed, for example, by mutation (see FIG. 21).

Signal peptides or small molecules comprising or mimicking co-activatorbinding domains are designed. Also, molecules capable of specificallyrecognizing and recruiting basal transcriptional proteins such as TAFIIproteins and/or RNA polymerases are designed as wild-type orsynthetically modified by non-natural amino acids and peptide stapling.Specifically, the KIX domain of CBP/p300 has two distinct binding sitestargeted by transcription factors to localize and retain theco-activator protein (see FIG. 24B). Suitable alpha-helical peptide “E”domains targeting these binding sites include:

p53 AD1: (SEQ ID NO: 45) Ac-LSQETFSDLWKLLPE-CONH₂ p53 AD2:(SEQ ID NO: 46) Ac-MLSPDDIEQWFTEDPG-CONH₂ MLL: (SEQ ID NO: 47)Ac-ILPSDIMDFVLKNTP-CONH₂ c-Jun: (SEQ ID NO: 48) Ac-LASPELERLIIQSSN-CONH₂HLTV-TAX: (SEQ ID NO: 49) Ac-YIDGFVIGSALQFLIPRLP-CONH₂ c-MYB:(SEQ ID NO: 50) Ac-KEKRIKELELLLMSTENELKG-CONH₂ pKID: (SEQ ID NO: 51)Ac-ILSRRPSYRKILNDLSSDAPG-CONH₂

Stapled “E” peptides derived from c-Myb are:

(SEQ ID NO: 52) Ac-KEKRIKELEL*LMS*ENELKG-CGNH₂ (SEQ ID NO: 53)Ac-KEKRIK*LEL*LMSTENELKG-CGNH₂ (SEQ ID NO: 54)Ac-KE*RIK*LELLLMSTENELKG-CGNH₂ (SEQ ID NO: 55)Ac-KEKRIK#LELLLM*TENELKG-CGNH₂ (SEQ ID NO: 73) K*KRI*ELELLLMSTENELKG(SEQ ID NO: 74) K*KRI*RLELLLMSTENELKG (SEQ ID NO: 75)KE*RIK*LELLLMSTENELKG (SEQ ID NO: 76) KR*RIK*LELLLMSTENELKG(SEQ ID NO: 77) KE*RIKELE*LLMSTENELKG (SEQ ID NO: 78)KE*RIKRLE*LLMSTENELKG (SEQ ID NO: 79) KR*RIKELE*LLMSTENELKG(SEQ ID NO: 80) KEKRIKELELLLMSTE*ELK*

Stapled “E” peptides derived from MLL:

(SEQ ID NO: 56) Ac-*ILP*DIMDFVLKNTP-CONH₂ (SEQ ID NO: 57)Ac-ILP*DIM*FVLKNTP-CONH₂ (SEQ ID NO: 58) Ac-ILPSDIM*FVL*NTP-CONH₂(SEQ ID NO: 59) Ac-ILPSDIMDFV*KNT*-CONH₂ (SEQ ID NO: 60)Ac-#ILPSDI*DFVLKNTP-CONH₂ (SEQ ID NO: 81) ILP*DIM*FVLKNT (SEQ ID NO: 82)ILP*RIM*FVLKNT (SEQ ID NO: 83) ILPSDIM*FVL*NT (SEQ ID NO: 84)ILPSRIM*FVL*NT

Stapled “E” peptides derived from p-KID (where any serine residues, inparticular Ser133, can be phosphorylated, as is present in the nativepKID:KIX interaction):

(SEQ ID NO: 61) Ac-ILSRRPSY*KIL*DLSSDAPG-CONH₂ (SEQ ID NO: 62)Ac-ILSRRPSYRKIL*DLS*DAPG-CONH₂ (SEQ ID NO: 63)Ac-ILSR*PSY*KILNDLSSDAPG-CONH₂ (SEQ ID NO: 64)Ac-ILSRRPSYR*ILN*LSSDAPG-CONH₂ (SEQ ID NO: 65)Ac-ILSRRP#YRKILN*LSSDAPG-CONH₂ (SEQ ID NO: 66)Ac-ILSRRPSYRKILNDLSSDAPG-CONH₂

-   -   Where “*” is the non-natural amino acid S5, and “#” is the        non-natural amino acid R8. In any arrangement, these amino acids        are cross-linked.

Example 13: General Transcription Factor Post-Translational Modificationby Tethered Effector Domains

Effector domains (E) are designed comprising peptides or small moleculescapable of binding and recruiting specific post-translational modifyingenzymes or complexes including kinases, acetyltransferases,phosphatases, glycotransferases, lipid transferases, and other enzymesknown to alter transcription factor function (see FIG. 22).

Example 14: Design and Synthesis of Bifunctional Stapled Peptides

Transcription factor targeting ligand, such as SAHM1, a designed stapledpeptide capable of binding the Notch:CSL transcription factor complex isdesigned as a targeting domain (A). For example, SAHM1:Ac-Bala-ERLRRRI*LCR*HHST-CONH₂ (SEQ ID NO:73), where “*” is thenon-natural amino acid S5, is designed, where SAHM1 is capable ofbinding the Notch:CSL transcription complex (WO 2008/061192,incorporated herein by reference in its entirety).

“E”—Effector domain capable of binding and recruiting cellular machineryto the transcription factor of interest is designed. The goal is tosynthesize a tethered form of “A” and “E” such that they areindependently functionally active to bind their targets. Through thetether, however, their functions are linked enacting the effects of theeffector protein on the TF of interest. Linker synthesis is carried outas outlined in FIG. 23.

Example 15: Stapled Repressive Domains

Stapled repressive domains were developed and analyzed based on effectordomains that associate with Sin3 (FIG. 24A). The Sin3 protein is anevolutionary conserved repressor that is part of a 1.2 MDa multi-proteinco-repressor complex associated with HDAC activity. The core subunits ofthe Sin3 complex include HDAC1, HDAC2, RbAp46/48, RBP1, SAPI30, BRMS1,SDS3, SAP30, and SAP18. Sin3 contains four conserved imperfect repeatsof 100 amino acids termed paired amphipathic helix (PAH) domains whichare protein-protein interaction modules. PAH1 is thought to interactwith Opi1, Pf1, NRSF, N-CoR, and SMRT. The PAH2 domain interactions forexample with Mad protein family members, Sp1-like repressor poteins,HBP1, Pf1, and yeast Ume6. The ability of the tumor suppressor Mad toinhibit cell proliferation and to repress transcription is dependent onan N-terminal N⁸IQMLLEAADYLE²⁰ domain named SID (Sin3 interactingdomain). In nuclear magnetic resonance experiments, Mad SID folds as anamphipathic helix and contacts the PAH2 domain of Sin3 which folds as afour-helix bundle (Brubaker, K. et al. Cell 103, 655-665 (2000)). A SIDconsensus sequences for Mad family members is thought to comprise thefollowing degenerate sequence: ΦZZΦΦXAAXXΦnXXn with X being anynon-proline residue, Φ being a bulky hydrophobic residue, and n beingnegatively charged residues (Guezennec et al. Nucl. Acid Res. 34(14):3929-3937 (2006)).

Peptides SID1 to SID9 were synthesized:

SIDLong(5-28): (SEQ ID NO: 85) VRMNIQMLLEAADYLERREREAEH SIDshort(5-24):(SEQ ID NO: 86) VRMNIQMLLEAADYLERRER Consensus: XXXΦZZΦΦXAAXXΦEX SID1:(SEQ ID NO: 87) pAla-ERLRRRI*MLL*AANYLER SID2: (SEQ ID NO: 88)pAla-VRRRI*MLL*AANYLER SID3: (SEQ ID NO: 89) pAla-VRRRIQRLL*AAN*LERSID4: (SEQ ID NO: 90) pAla-VRMNIQMLLQAANR*ERR*R SIDS: (SEQ ID NO: 91)pAla-VRRRIQMLLEAANK*ERR*R SID6: (SEQ ID NO: 92)pAla-VRMNIQMLLQAANRLERR*REA*H SID7: (SEQ ID NO: 93)pAla-VRRRIQMLLEAANKLERR*REA*H SID8: (SEQ ID NO: 94)pAla-VRMNIQMLL*AAN*LER SID9: (SEQ ID NO: 95) pAla-VRMNI*MLL*AANYLER,where “*” is the non-natural amino acid S5, and these amino acids arecross-linked. FIG. 25B shows a sample fluorescent polarizationexperiment data for SID2 and SID5 as compared to wild type SID used todetermine dissociation constants (K_(D)). Sin3 binding assays wereperformed by incubating FITC-SID peptides (10 nM) with serial dilutionsof Sin3 in a buffer of 50 mM NaCl, 1 mM DTT, 10 mM Tris pH 7.4.Dilutions and incubations were made in 384-well, black flat-bottomplates (Corning) to a total volume of 100 μL and incubated for 2 hours.Polarization was measured on a Spectramax-M5 multi-label plate readerwith λ_(ex)=485 nm and λ_(em)=525 nm. Polarization was calculatedaccording to the standard equation: P=(V−H)/(V+H), where P=polarization,V=vertical emission intensity and H=horizontal emission intensity. K_(d)values were determined by fitting data to a variable-slope sigmoidalbinding curve using Kaleidagraph.

FIG. 25C shows confocal microscopy of Hela cells treated withFITC-conjugated SID-series peptides. SID2 and SID5 reveal robustcellular penetration. HeLa cells were grown on chamber slides overnight.10 mM FITC-SID peptides in DMSO stock solutions were diluted in cellmedia to a final concentration of 10 μM, along with a 10 μM DMSOcontrol. Cells were incubated in peptide/vehicle solutions at 37° C. for6 hours, then washed thoroughly with media and PBS, and fixed with 4%paraformaldehyde. Slides were stained with Vectashield™ Hardset withDAPI. Images were taken with a Zeiss 710 confocal microscope.

Example 16: Covalent Conjugation Strategies for Bifunctional StapledPeptides

FIG. 27 provides an overview of exemplary conjugation strategies ofassociating two stapled peptides via chemical linkers (FIG. 27). Forexample, thiol (—SH) groups and maleimide groups were used as reactivegroups to generate thiol-maleimide conjugates. The groups were reactedin a 4:1 PBS/CH₃CN solution at pH 7.4 (FIG. 30). Azide (N₃) groups werereacted with alkyne groups using a Cu^(I/II) catalyst and a reducingagent in organic or aqueous solvent to obtain 1,4- or 1,5-triazolemoieties. Alkyne/azide reactive groups of various length andconfiguration may be used.

Resin-coupled, maleimide-containing stapled peptides were generatedreacting the Mmt-protected lysine residue of the stapled peptide asolution containing 1:4:95 (vol/vol) trifluoroacetic acid (TFA):triisopropyl silane (TIS): dichloromethane (DCM). The deprotectedamine-containing stapled peptides were coupled with NHS-Maleimide inDMF/diisopropylethyl amine and the resulting maleimide-containingstapled peptides were cleaved off the resin using standard peptidecleavage and deprotection in a solution containing 2.5:2.5:95 (vol/vol)water:TIS:TFA (FIG. 28).

Thiol-containing stapled peptides were generated from resin-coupled,cysteines-containing, protected stapled peptides. Peptide release fromthe resin was accomplished using standard peptide cleavage anddeprotection in a solution containing 2.5:2.5:95 (vol/vol) water:TIS:TFA(FIG. 29).

FIG. 31 shows the mass spectrum of a thiol-containing stapled peptide(upper panel), a maleimide-containing stapled peptide (middle panel),and a reacted conjugated bifunctional stapled peptide (lower panel).FIG. 32 shows a mass spectrum of the HPLC-purified conjugatedbifunctional thiol-maleimide stapled peptide.

1-111. (canceled)
 112. A bifunctional stapled peptide comprising aneffector domain selected from: (SEQ ID NO: 36) Ac-LSQETFS*LWK*LPE-CONH₂;(SEQ ID NO: 37) Ac-QSQQTF#NLWRKK*QN-CONH₂; (SEQ ID NO: 38)Ac-QSQQTF*NLW*KKQN-CONH₂; and (SEQ ID NO: 39) Ac-LSQNTFS*LWK*LPQ-CONH₂,

where “*” is the non-natural amino acid S5, and “#” is the non-naturalamino acid R8.
 113. A bifunctional stapled peptide comprising aneffector domain selected from: (SEQ ID NO: 67)Ac-VRIVINIQMLLEA*DYL*RRER-CONH₂; (SEQ ID NO: 68)Ac-VRIVINIQM*LEA*DYLERRER-CONH₂; (SEQ ID NO: 69)Ac-VRIVINIQML#EAADYL*RRER-CONH₂; and (SEQ ID NO: 70)Ac-VRIVI*IQM*LEAADYLERRER-CONH₂,

where “*” is the non-natural amino acid S5, and “#” is the non-naturalamino acid R8.
 114. A bifunctional stapled peptide comprising aneffector domain selected from: (SEQ ID NO: 52)Ac-KEKRIKELEL*LMS*ENELKG-CONH₂; (SEQ ID NO: 53)Ac-KEKRIK*LEL*LMSTENELKG-CONH₂; (SEQ ID NO: 54)Ac-KE*RIK*LELLLMSTENELKG-CONH₂; (SEQ ID NO: 55)Ac-KEKRIK#LELLLM*TENELKG-CONH₂; (SEQ ID NO: 73) K*KRI*ELELLLMSTENELKG;(SEQ ID NO: 74) K*KRI*RLELLLMSTENELKG; (SEQ ID NO: 75)KE*RIK*LELLLMSTENELKG; (SEQ ID NO: 76) KR*RIK*LELLLMSTENELKG;(SEQ ID NO: 77) KE*RIKELE*LLMSTENELKG; (SEQ ID NO: 78)KE*RIKRLE*LLMSTENELKG; (SEQ ID NO: 79) KR*RIKELE*LLMSTENELKG;(SEQ ID NO: 80) KEKRIKELELLLMSTE*ELK*; (SEQ ID NO: 56)Ac-*ILP*DIMDFVLKNTP-CONH₂; (SEQ ID NO: 57) Ac-ILP*DEVI*FVLKNTP-CONH₂;(SEQ ID NO: 58) Ac-ILPSDIM*FVL*NTP-CONH₂; (SEQ ID NO: 59)Ac-ILPSDIMDFV*KNT*-CONH₂; (SEQ ID NO: 60) Ac-#ILPSDI*DFVLKNTP-CONH₂;(SEQ ID NO: 81) ILP*DEVI*FVLKNT; (SEQ ID NO: 82) ILP*RIM*FVLKNT;(SEQ ID NO: 83) ILPSDIM*FVL*NT; (SEQ ID NO: 84) ILPSRIM*FVL*NT;(SEQ ID NO: 61) Ac-ILSRRPSY*KIL*DLSSDAPG-CONH₂; (SEQ ID NO: 62)Ac-ILSRRPSYRKIL*DLS*DAPG-CONH₂; (SEQ ID NO: 63)Ac-ILSR*PSY*KILNDLSSDAPG-CONH₂; (SEQ ID NO: 64)Ac-ILSRRPSYR*ILN*LSSDAPG-CONH₂; (SEQ ID NO: 65)Ac-ILSRRP#YRKILN*LSSDAPG-CONH₂; and (SEQ ID NO: 66)Ac-ILSRRPSYRKILNDLSSDAPG-CONH₂,

where “*” is the non-natural amino acid S5, and “#” is the non-naturalamino acid R8.
 115. A pharmaceutical composition comprising a peptide ofclaim 112 or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable excipient.
 116. A pharmaceutical compositioncomprising a peptide of claim 113 or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable excipient.
 117. Apharmaceutical composition comprising a peptide of claim 114 or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient.
 118. A method of treating a disease, disorder, orcondition in a subject, comprising administering a therapeuticallyeffective amount of a peptide of claim 112 to a subject in need thereof.119. The method of claim 118, wherein the disease, disorder, orcondition is cancer, hematopoietic neoplastic disorders, proliferativebreast disease, proliferative disorders of the lung, proliferativedisorders of the colon, proliferative disorders of the liver, andproliferative disorders of the ovary.
 120. A method of treating adisease, disorder, or condition in a subject, comprising administering atherapeutically effective amount of a peptide of claim 113 to a subjectin need thereof.
 121. The method of claim 120, wherein the disease,disorder, or condition is cancer, hematopoietic neoplastic disorders,proliferative breast disease, proliferative disorders of the lung,proliferative disorders of the colon, proliferative disorders of theliver, and proliferative disorders of the ovary.
 122. A method oftreating a disease, disorder, or condition in a subject, comprisingadministering a therapeutically effective amount of a peptide of claim114 to a subject in need thereof.
 123. The method of claim 122, whereinthe disease, disorder, or condition is cancer, hematopoietic neoplasticdisorders, proliferative breast disease, proliferative disorders of thelung, proliferative disorders of the colon, proliferative disorders ofthe liver, and proliferative disorders of the ovary.